Codon optimized REP1 genes and uses thereof

ABSTRACT

The present disclosure provides codon optimized nucleotide sequences encoding human REP1, vectors, and host cells comprising codon optimized REP1 sequences, and methods of treating retinal disorders such as choroideremia comprising administering to the subject a codon optimized sequence encoding human REP1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/463,262, filed Aug. 31, 2021, which claims the benefit of U.S.Provisional Patent Application Ser. No. 63/073,837, filed Sep. 2, 2020,the full disclosure of each of which is incorporated herein byreference.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled“090400-5011-US-Sequence-Listing” created on or about Aug. 31, 2021,with a file size of about 27 KB contains the sequence listing for thisapplication and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Choroideremia is a rare, X-linked recessive form of hereditary retinaldegeneration that affects roughly 1 in 50,000 males. The disease causesa gradual loss of vision, starting with childhood night blindness,followed by peripheral vision loss and progressing to loss of centralvision later in life. Progression continues throughout the individual'slife, but both the rate of change and the degree of visual loss arevariable among those affected, even within the same family.

Choroideremia is caused by a loss-of-function mutation in the CHM genewhich encodes Rab escort protein-1 (REP1), a protein involved in lipidmodification of Rab proteins. While the complete mechanism of disease isnot fully understood, the lack of a functional protein in the retinaresults in cell death and the gradual deterioration of the retinalpigment epithelium (RPE), photoreceptors and the choroid.

Although there are currently no approved treatments for choroideremia,several preclinical studies support the use of wild type cDNA of CHM torescue the choroideremia disease phenotype. However, suboptimalexpression level of the wild type sequence in human photoreceptors andRPE are challenges to gene therapy approaches to treat choroideremia.

SUMMARY OF THE INVENTION

Disclosed are codon optimized nucleic acid molecules encoding a humanRab escort protein-1 (REP1) protein. In one aspect, the disclosureprovides a nucleic acid comprising the nucleotide sequence of SEQ IDNO:1 or a nucleic acid comprising a nucleotide sequence at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the nucleotide sequence of SEQ ID NO:1 and which encodes ahuman REP1 polypeptide having the amino acid sequence of SEQ ID NO:2. Insome embodiments, a nucleic acid comprising or consisting of thenucleotide sequence of SEQ ID NO:1 is provided. In related embodiments,the nucleic acid is expressed at a higher level compared with the levelof expression of a wild type CHM nucleic acid sequence (e.g. SEQ IDNO:3) in an otherwise identical cell.

In some aspects, a codon optimized nucleic acid molecule as hereindescribed has a human codon adaptation index that is increased relativeto that of the wild type CHM cDNA (GenBank Accession No. NM_000390.4;SEQ ID NO:3). In some embodiments, the codon optimized nucleic acidmolecule has a human codon adaptation index of at least about 0.9, atleast about 0.92, or at least about 0.94.

In certain embodiments, the nucleic acid contains a higher percentage ofG/C nucleotides compared to the percentage of G/C nucleotides in SEQ IDNO:3. In other embodiments, the nucleic acid contains a percentage ofG/C nucleotides that is at least about 55%, at least about 57.5%, atleast about 60% or at least about 61%. In some aspects, the nucleic acidcontains a percentage of G/C nucleotides that is from about 55% to about70%, from about 57.5% to about 70% or from about 61% to about 70%.

In other embodiments, the nucleic acid comprises one or more optimizedparameters relative to SEQ ID NO:3: frequency of optimal codons;reduction in maximum length of direct repeat sequences; removal ofrestriction enzymes, including without limitation, removal ofBglll(AGATCT); removal of CIS-acting elements, including withoutlimitation, and removal of destabilizing (ATTTA) elements.

In another embodiment, the nucleic acid is operatively linked to atleast one transcription control sequence, preferably a transcriptioncontrol sequence that is heterologous to the nucleic acid. In someaspects, the transcription control sequence is a cell- ortissue-specific promoter that results in cell-specific expression of thenucleic acid e.g. in photoreceptor cells such as vitelliform maculardystrophy 2 promoter which is selectively expressed in the RPE. In otheraspects, the transcription control sequence is a constitutive promoterthat results in similar expression level of the nucleic acid in manycell types (e.g. a CAG, CBA (chicken beta actin), CMV, or PGK promoter).In preferred embodiments, the transcription control sequence comprises aCAG promoter comprising (i) the cytomegalovirus (CMV) early enhancerelement, (ii) the promoter, first exon and first intron of chickenbeta-actin gene and (iii) the splice acceptor of the rabbit beta-globingene as described in Miyazaki et al., Gene 79(2):269-77 (1989). In aparticularly preferred embodiment, the CAG promoter comprises thesequence of SEQ ID NO:4 or comprises a sequence at least 95%, at least96%, at least 97%, at least 98% or at least 99% identical thereto:

(SEQ ID NO: 4) ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCAGTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCGCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGOGGTGTGGGCGCGTCGGTGGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGGGGCGGCCCCCGGAGGGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACGCCCTGTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGGCTTCTTCTTTTTCCTACAG 

The transcription control sequence may also comprise one or moreelements downstream of the REP1 coding sequence such as a Woodchuckhepatitis virus posttranscriptional regulatory element (WPRE), which hasbeen shown to enhance AAV transgene expression in the retina. In relatedembodiments, provided herein is an expression cassette comprising anucleic acid comprising the nucleotide sequence of SEQ ID NO:1, or anucleotide sequence at least 90% identical thereto, operably linked toan expression control sequence.

In related embodiments, provided herein is a vector comprising a nucleicacid comprising the nucleotide sequence of SEQ ID NO:1, or a nucleotidesequence at least 90% identical thereto. In preferred embodiments, thevector is a recombinant adeno-associated (rAAV) expression vector. Insome embodiments, the rAAV vector comprises a native capsid (e.g. acapsid of AAV serotype 2, AAV serotype 4, AAV serotype 5 or AAV serotype8). In other embodiments, the rAAV vector comprises a capsid that ismodified (e.g. comprises one or more peptide insertions and/or one ormore amino acid substitutions (e.g. tyrosine to phenylalanine) and/oramino acid insertions or amino acid deletions) relative to a native AAVcapsid (e.g. comprising one or more modifications relative to an AAVcapsid of serotype 2, 4, 5 or 8).

In another embodiment, provided herein is a host cell comprising anucleic acid comprising the nucleotide sequence of SEQ ID NO:1, or anucleotide sequence at least 90% identical thereto. In some aspects, thehost cell is a mammalian cell, including without limitation, a CHO cell,an HEK293 cell, a HeLa cell, a BHK21 cell, a Vero cell or a V27 cell. Inrelated aspects, the host cell is selected from a CHO cell, an HEK293cell, an HEK293T cell, a HeLa cell, a BHK21 cell and a Vero cell. Inother aspects, the host cell is a photoreceptor cell (e.g. rods; cones),a retinal ganglion cell (RGC), a glial cell (e.g. a Muller glial cell, amicroglial cell), a bipolar cell, an amacrine cell, a horizontal cell,or a retinal pigmented epithelium (RPE) cell. In related embodiments,the disclosure provides a method of increasing expression of apolypeptide of SEQ ID NO: 2 comprising culturing the host cell underconditions whereby a polypeptide of SEQ ID NO: 2 is expressed by thenucleic acid molecule, wherein the expression of the polypeptide isincreased relative to a host cell cultured under the same conditionscomprising a reference nucleic acid comprising the nucleotide sequenceof SEQ ID NO:3 (comparator sequence).

In another embodiment, the disclosure provides a method of increasingexpression of a polypeptide of SEQ ID NO: 2 in a human subjectcomprising administering to the subject an isolated nucleic acidmolecule comprising a nucleotide sequence at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identical, or 100% identical to the nucleotide sequence of SEQ ID NO:1and which encodes a polypeptide having the amino acid sequence of SEQ IDNO:2 or a vector comprising such a nucleotide sequence, wherein theexpression of the polypeptide is increased relative to a referencenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3(comparator sequence) or a vector comprising the reference nucleic acidmolecule.

In some embodiments, the disclosure provides a method of treating anocular disorder associated with insufficient REP1 activity in a humansubject comprising administering to the subject a nucleic acid moleculeor a vector disclosed herein. In some embodiments, the retinal disorderis choroideremia.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D FIGS. 1A and 1B illustrate immunocytochemical analysis ofiPSCs derived from choroideremia patients CHM1 (FIG. 1A) and CHM2 (FIG.1B) using antibodies against pluripotent transcription factors NANOG,OCT4 and SOX2. FIGS. 1C and 1D illustrate representative images of CHM1(FIG. 1C) and CHM2 (FIG. 1D) cultures randomly differentiated intoectodermal, mesodermal and endodermal cell lineages as indicted byexpression of TUJ1, α-smooth muscle actin (ASMA) and Hepatocyte NuclearFactor 4 Alpha (HNF4A).

FIG. 2A-C FIGS. 2A and 2B illustrate immunocytochemical analysis of RPEcells derived from iPSCs derived from choroideremia patients CHM1 (FIG.2A) and CHM2 (FIG. 2B). The RPE phenotype after 45 days ofdifferentiation and maturation by ICC showed proper RPE transcriptionalfactor expression of Melanogenesis Associated Transcription Factor(MITF) and Orthodenticle Homeobox 2 (OTX2), expression of mature RPEcell marker RPE65, and expression of tight junction marker ZonulaOccludens (ZO-1). The nuclei were counterstained with DAPI for CHM1images. Scale bar=50 μm. FIG. 2C is a graph illustrating that CHM1 RPEand CHM2 RPE phagocytose at similar levels to wild type (WT) RPE. Inaddition, phagocytosis occurs through the known mechanism in vivo, αVβ5integrin binding, as seen by a decrease in phagocytosis following αVβ5inhibition. n=3 for quantitative measurements; Error bars±S.D.; *p<0.05,compared to the “No ROS” condition; two-tailed t-test.

FIGS. 3A-B FIG. 3A: Western blot images are shown illustrating REP1 andhousekeeping protein GADPH levels in CHM1 RPE or normal iPSC-derived RPEcells following transduction with recombinant AAV virions carrying thecodon optimized REP1 gene or carrying the unmodified REP1 gene, in eachcase driven by a CAG promoter. The codon optimized REP1 showedsignificantly higher levels of protein expression. FIG. 3B: bandintensity was quantified and graphed as a ratio over GAPDH. n=3 forquantitative measurements; Error bars±S.D.; *p<0.05, compared to theWT-REP1; two-tailed t-test.

FIG. 4 Schematic illustration of the functional prenylation assayshowing required components, including the biotinylated prenyl groupserving as the readout of the biochemical assay. Normal RPE cells withfunctional REP1 protein successfully facilitate the prenylation ofRab27A GTPase, leading to the incorporation of the biotin groups. In CHMRPE, cells lack REP1 protein, causing accumulation of unprenylatedRab27a GTPase protein.

FIGS. 5A-5D Transduction of CHM1 and CHM2 RPE with rAAV virionscomprising codon optimized REP1 of SEQ ID NO:1 under the control of aCAG promoter restored prenylation of Rab27a GTPase. FIGS. 5A and 5B: Gelimages illustrating the level of REP1 protein by Western blot analysisand incorporation of a biotinylated prenyl donor as a measure ofprenylation in cell lysates, in transduced and untransduced CHM1 (FIG.5A) and CHM2 (FIG. 5B) RPE cells (compared to normal iPSC-derived RPEcells). FIGS. 5C and 5D: Band intensity was quantified and depicted inbar graphs as biotinylated Rab27a GTPase relative to the housekeepingprotein GADPH, in CHM1 (FIG. 5C) and CHM2 (FIG. 5D) RPE cells. n=3 forquantitative measurements; Error bars±S.D.; *p<0.05, compared to theuntreated CHM RPE; two-tailed t-test.

FIGS. 6A-6C FIG. 6A: Immunostaining of CHM1 RPE with anti-REP1 andanti-RAB27A antibodies illustrates that CHM1 RPE lacked proper membranelocalization of Rab27a GTPase. FIGS. 6B and 6C: Delivery of codonoptimized REP1 of SEQ ID NO:1 by rAAV vector, corrected Rab27 GTPasemembrane trafficking in CHM1 RPE (FIG. 6B), and restored localization toa control RPE phenotype (FIG. 6C).

FIG. 7 : DNA alignment of the optimized region of SEQ ID NO:1 withnative REP1 of SEQ ID NO:3.

FIG. 8 is a schematic of the transgene cassette contained within therAAV described in Example 2 below. The transgene cassette comprises a5′AAV2 ITR, a CAG Promoter, a Codon Optimized Human CHM cDNA of SEQ IDNO:1, an SV40 Polyadenylation Signal, and a 3′ AAV2 ITR and has thenucleotide sequence of SEQ ID NO:5.

FIG. 9 illustrates safety of 4D-110 (comprising the transgene cassetteshown in FIG. 8 and a capsid protein of SEQ ID NO:9) followingintravitreal administration to non-human primates through quantificationof ocular inflammation, as assessed by aqueous flare, aqueous cells, andvitreous cells. Ophthalmoscopic signs of transient mild ocularinflammation were observed at the high dose. These changes responded toan increase in the systemic steroid treatment. There were no adversefindings considered related to 4D-110. IOP values were within normallimits for all animals at the different examination intervals. ERGvalues and OCT images including macular morphology were also withinnormal limits.

FIG. 10 illustrates vector genome biodistribution in selected retinal,ocular, and non-ocular tissues, as measured by qPCR at 3 necropsytimepoints in NHPs intravitreally administered 4D-110. LOD=lower limitof detection; all samples “BLOD” graphed at LOD value for visualizationpurposes.

FIG. 11 illustrates REP1 transgene mRNA expression in selected retinal,ocular, and non-ocular tissues, as measured by RT-qPCR at 3 necropsytimepoints in NHPs intravitreally administered 4D-110. LOD=lower limitof detection; all samples “BLOD” graphed at LOD value for visualizationpurposes.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “codon adaptation index,” as used herein, refers to a measure of codonusage bias. A codon adaptation index (CAI) measures the deviation of agiven protein coding gene sequence with respect to a reference set ofgenes (Sharp P M and Li W H, Nucleic Acids Res. 15(3):1281-95 (1987)).CAI is calculated by determining the geometric mean of the weightassociated to each codon over the length of the gene sequence (measuredin codons):

$\begin{matrix}{{{CAI} = {\exp\left( {1\text{/}L{\sum\limits_{l = 1}^{L}\;{\ln\left( {w_{1}(l)} \right)}}} \right)}},} & (I)\end{matrix}$For each amino acid, the weight of each of its codons, in CAI, iscomputed as the ratio between the observed frequency of the codon (fi)and the frequency of the synonymous codon (fj) for that amino acid:

$\begin{matrix}{w_{i} = {{\frac{f_{i}}{\max\left( f_{j} \right)}\mspace{14mu}{ij}} \in \left\lbrack {{synonymous}\mspace{14mu}{codons}\mspace{14mu}{for}\mspace{14mu}{amino}\mspace{14mu}{acid}} \right\rbrack}} & ({II})\end{matrix}$

The term “isolated” designates a biological material (cell, nucleic acidor protein) that has been removed from its original environment (theenvironment in which it is naturally present). For example, apolynucleotide present in the natural state in a plant or an animal isnot isolated, however the same polynucleotide separated from theadjacent nucleic acids in which it is naturally present, is considered“isolated.”

The term “4D-110” refers to a recombinant AAV particle comprising (i) acapsid protein comprising the amino acid sequence of SEQ ID NO:9 and aheterologous nucleic acid comprising the nucleotide sequence of SEQ IDNO:5.

The term “R100” refers to a variant AAV capsid protein comprising theamino acid sequence of SEQ ID NO:9.

The term “having” as used herein is equivalent to the term “comprising”and is intended to be open-ended allowing additional elements.

As used herein, a “coding region” or “coding sequence” is a portion ofpolynucleotide which consists of codons translatable into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is typically not translatedinto an amino acid, it can be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, and the like, are not partof a coding region. The boundaries of a coding region are typicallydetermined by a start codon at the 5′ terminus, encoding the aminoterminus of the resultant polypeptide, and a translation stop codon atthe 3′ terminus, encoding the carboxyl terminus of the resultingpolypeptide. Two or more coding regions can be present in a singlepolynucleotide construct, e.g., on a single vector, or in separatepolynucleotide constructs, e.g., on separate (different) vectors. Itfollows, then that a single vector can contain just a single codingregion, or comprise two or more coding regions.

As used herein, the term “regulatory region” refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding region, and whichinfluence the transcription, RNA processing, stability, or translationof the associated coding region. Regulatory regions can includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures. If a coding region is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

As used herein, the term “nucleic acid” is interchangeable with“polynucleotide” or “nucleic acid molecule” and a polymer of nucleotidesis intended.

A polynucleotide which encodes a gene product, e.g., a polypeptide, caninclude a promoter and/or other transcription or translation controlelements operably associated with one or more coding regions. In anoperable association a coding region for a gene product, e.g., apolypeptide, is associated with one or more regulatory regions in such away as to place expression of the gene product under the influence orcontrol of the regulatory region(s). For example, a coding region and apromoter are “operably associated” if induction of promoter functionresults in the transcription of mRNA encoding the gene product encodedby the coding region, and if the nature of the linkage between thepromoter and the coding region does not interfere with the ability ofthe promoter to direct the expression of the gene product or interferewith the ability of the DNA template to be transcribed. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can also be operably associated with a coding region to direct geneproduct expression.

“Transcriptional control sequences” refer to DNA regulatory sequences,such as promoters, enhancers, terminators, and the like, that providefor the expression of a coding sequence in a host cell. A variety oftranscription control regions are known to those skilled in the art.These include, without limitation, transcription control regions whichfunction in vertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (the immediate early promoter,in conjunction with intron-A), simian virus 40 (the early promoter), andretroviruses (such as Rous sarcoma virus). Other transcription controlregions include those derived from vertebrate genes such as actin, heatshock protein, bovine growth hormone and rabbit beta-globin, as well asother sequences capable of controlling gene expression in eukaryoticcells. Additional suitable transcription control regions includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

The term “expression” as used herein refers to a process by which apolynucleotide produces a gene product, for example, an RNA or apolypeptide. It includes without limitation transcription of thepolynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), smallhairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNAproduct, and the translation of an mRNA into a polypeptide. Expressionproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation or splicing, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, or proteolytic cleavage.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector can be a replicon to whichanother nucleic acid segment can be attached so as to bring about thereplication of the attached segment. The term “vector” includes bothviral and nonviral vehicles for introducing the nucleic acid into a cellin vitro, ex vivo or in vivo. A large number of vectors are known andused in the art including, for example, plasmids, modified eukaryoticviruses, or modified bacterial viruses. Insertion, of a polynucleotideinto a suitable vector can be accomplished by ligating the appropriatepolynucleotide fragments into a chosen vector that has complementarycohesive termini.

Vectors can be engineered to encode selectable markers or reporters thatprovide for the selection or identification of cells that haveincorporated the vector. Expression of selectable markers or reportersallows identification and/or selection of host cells that incorporateand express other coding regions contained on the vector. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like. Examples of reporters knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ),-glucuronidase (Gus), and the like. Selectable markers can also beconsidered to be reporters.

Eukaryotic viral vectors that can be used include, but are not limitedto, adenovirus vectors, retrovirus vectors, adeno-associated virusvectors, poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, orherpesvirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers.

“Promoter” and “promoter sequence” are used interchangeably and refer toa DNA sequence capable of controlling the expression of a codingsequence or functional RNA. In general, a coding sequence is located 3′to a promoter sequence. Promoters can be derived in their entirety froma native gene, or be composed of different elements derived fromdifferent promoters found in nature, or even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentpromoters can direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental or physiological conditions. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters.” Promoters that cause agene to be expressed in a specific cell type are commonly referred to as“cell-specific promoters” or “tissue-specific promoters.” Promoters thatcause a gene to be expressed at a specific stage of development or celldifferentiation are commonly referred to as “developmentally-specificpromoters” or “cell differentiation-specific promoters.” Promoters thatare induced and cause a gene to be expressed following exposure ortreatment of the cell with an agent, biological molecule, chemical,ligand, light, or the like that induces the promoter are commonlyreferred to as “inducible promoters” or “regulatable promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths can have identical promoter activity.

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements can be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wis., USA. Other techniques for alignmentare described in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc. Of particular interest are alignment programs that permit gaps inthe sequence. The Smith-Waterman is one type of algorithm that permitsgaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997).Also, the GAP program using the Needleman and Wunsch alignment methodcan be utilized to align sequences. See J. Mol. Biol. 48: 443-453(1970).

In one embodiment, the present invention provides a modified nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide of SEQ ID NO:2 (human REP1), wherein the nucleic acidsequence has been codon optimized. In another embodiment, the startingnucleic acid sequence that encodes a polypeptide of SEQ ID NO:2 and thatis subject to codon optimization has the nucleotide sequence set forthas SEQ ID NO:3. In preferred embodiments, the sequence that encodes apolypeptide of SEQ ID NO:2 is codon optimized for human expression. SEQID NO:1 is a codon optimized version of SEQ ID NO:3, optimized for humanexpression:

(SEQ ID NO: 1) ATGGCTGATACACTGCCTTCTGAGTTTGATGTGATCGTGATTGGAACTGGACTGCCTGAGAGTATTATTGCTGCTGCTTGTAGTAGAAGCGGCCGGAGAGTGCTGCACGTGGACAGCAGATCCTACTATGGCGGCAACTGGGCCTCTTTCAGCTTTTCCGGCCTGCTGAGCTGGCTGAAGGAGTACCAGGAGAACTCCGACATCGTGTCTGATAGCCCCGTGTGGCAGGACCAGATCCTGGAGAATGAGGAGGCCATCGCCCTGTCCAGGAAGGATAAGACCATCCAGCACGTGGAGGTGTTCTGCTATGCCAGCCAGGACCTGCACGAGGATGTGGAGGAGGCAGGCGCCGTGCAGAAGAACCACGGCCTGGTGACCTCCGCCAATTCTACAGAGGCCGCCGACTCCGCCTTTCTGCCTACCGAGGATGAGTCCCTGTCTACAATGTCTTGTGAGATGCTGACCGAGCAGACACCTAGCTCCGATCCAGAGAACGCCCTGGAGGTCAATGGCGCCGAGGTGACCGGCGAGAAGGAGAACCACTGCGACGATAAGACCTGCGTGCCAAGCACATCCGCCGAGGACATGTCCGAGAACGTGCCTATCGCCGAGGATACCACAGAGCAGCCAAAGAAGAATCGCATCACATACAGCCAGATCATCAAGGAGGGCAGGCGCTTCAATATCGACCTGGTGTCTAAGCTGCTGTACAGCCGGGGCCTGCTGATCGATCTGCTGATCAAGAGCAACGTGTGCCGCTATGCCGAGTTCAAGAATATCACCAGAATCCTGGCCTTTCGGGAGGGAAGAGTGGAGCAGGTGCCCTGCAGCAGAGCCGACGTGTTCAACTCCAAGCAGCTGACAATGGTGGAGAAGAGGATGCTGATGAAGTTCCTGACATTTTGTATGGAGTACGAGAAGTATCCAGATGAGTACAAGGGCTATGAGGAGATCACCTTTTACGAGTATCTGAAGACCCAGAAGCTGACACCCAATCTGCAGTACATCGTGATGCACTCCATCGCCATGACCTCTGAGACAGCCTCTAGCACCATCGACGGCCTGAAGGCCACAAAGAACTTCCTGCACTGCCTGGGCCGGTACGGCAATACACCCTTCCTGTTTCCTCTGTATGGCCAGGGCGAGCTGCCCCAGTGCTTCTGTAGAATGTGCGCCGTGTTTGGCGGCATCTATTGCCTGAOGCACTCTGTGCAGTGTCTGGTGGTGGACAAGGAGAGCCGCAAGTGTAAGGCCATCATCGATCAGTTTGGCCAGCGGATCATCTCTCAGCACTTCCTGGTGGAGGACAGCTACTTTCCTGAGAACATGTGCTCGAGGGTGCAGTATCGCCAGATCAGCCGGGCGGTGCTGATCACCGATAGATCCGTGCTGAAGACAGACAGCGATCAGCAGATCAGCATCCTGACCGTGCGAGCAGAGGAGCCAGGCACCTTCGCCGTGAGAGTGATCGAGCTGTGCTCCTCTACCATGACATGTATGAAGGGCACCTAGCTGGTGCACCTGACCTGCACAAGCTCCAAGACAGCGCGCGAGGACCTGGAGAGCGTGGTGCAGAAGCTGTTCGTGCCCTACACCGAGATGGAGATCGAGAACGAGCAGGTGGAGAAGCCTAGAATCCTGTGGGCCCTGTACTTCAACATGAGAGACTCTAGCGATATCTCTAGGAGCTGTTACAACGATCTGCCCTCTAACGTGTACGTGTGCAGCGGACCTGACTGTGGCCTGGGAAAGGATAATGCCGTGAAGCAGGCCGAGACACTGTTCCAGGAGATTTGCCCTAACGAGGACTTTTGTCCCCCTCCACCCAATCCAGAQGATATCATCCTGGACGGCGATTCCCTGCAGCCAGAGGCCTCTGAGTCCTCTGCCATCCCCGAGGCCAATAGCGAAACATTCAAAGAAAGCACAAATCTGGGAAACCTGGAAGAAAGTAGTGAGT AA

In some embodiments, a codon-optimized sequence encoding human REP1 isprovided lacking the TAA stop codon of SEQ ID NO:1 (i.e. consisting ofnucleotides 1-1959 of SEQ ID NO:1).

In one aspect, the disclosure provides a polynucleotide comprising thenucleotide sequence of SEQ ID NO:1 or polynucleotide comprising anucleotide sequence at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to the nucleotide sequenceof SEQ ID NO:1 and which encodes a human REP1 polypeptide having theamino acid sequence of SEQ ID NO:2:

(SEQ ID NO: 2) MADTLPSEFDVIVIGTGPLPESIIAAACSRSGRRVLHVDSRSYYGGNWASFSFSGLLSWLKEYQENSDIVSDSPVWQDQILENEEIALSRKDKTIQHVEVFCYASQDLHEDVEEAGLQKNHALVTSANSTEAADSAFLPTEDESLSTMSCEMLTEQTPSSDPENALEVNGAEVTGEKENHCDDKTCVPSTSAEDMSENVPIAEDTTEQPKKNRITYSQIIKEGRRFNIDLVSKLLYSRGLLIDLLIKSNVSRYAEFKNITRILAFREGRVEQVPCSRADVFNSKQLTMVEKRMLMKFLTFCMEYEKYPDEYKGYEEITFYEYLKTQKLTPNLQYIVMHSIAMTSETASSTIDGLKATKNFLHCLGRYGNTPFLFPLYGQGELPQCFCRMCAVFGGIYCLRHSVQCLVVDKESRKCKAIIDQFGQRIISEHFLVEDSYFPENMCSRVQYRQISRAVLITDRSVLKTDSDQQISILTVPAEEPGTFAVRVIELCSSTMTCMKGTYVHLTCTSSKTAREDLESVVQKLFVPYTEMEIENEQVEKPRILWALYFNMRDSSDISRSCYNDLPSNVYVCSGPDCGLGNDNAVKQAETLFQEICPNEDFCPPPPNPEDIILDGDSLQPEASESSAIPEANSETF KESTNLGNLEESSE

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

Deviations in the nucleotide sequence that comprises the codons encodingthe amino acids of, any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE-US-00001 TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F)TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y)TGC TTA Leu (L) TCA Ser (S) TAA Stop TGA Stop TTG Leu (L) TCG Ser (S)TAG Stop TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R)CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P)CAA Gin (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG Gin (Q) CGG Arg (R)A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr(T) AAC Asn (N) AGC Ser (S) ATA Ile (1) ACA Thr (T) AAA Lys (K) AGA Arg(R) ATG Met (M) ACG Thr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCTAla (A) GAT Asp (D) GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGCGly (G) GTA Val (V) GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCGAla (A) GAG Glu (E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference, or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, the relative frequencies of codonusage have been calculated. Codon usage tables are available, forexample, at the “Codon Usage Database” available on the world wide webat kazusa.or.jp/codon/(visited Jun. 18, 2012). See Nakamura, Y., et al.Nucl. Acids Res. 28:292 (2000).

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs can be used to calculate an optimal sequence.

Non-Viral Vectors

In some embodiments, a non-viral vector (e.g. an expression plasmid)comprising a modified nucleic acid as herein described is provided.Preferably, the non-viral vector is a plasmid comprising a nucleic acidsequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto.

Viral Vectors

In preferred embodiments, a viral vector comprising a modified (codonoptimized) nucleic acid as herein described is provided. Preferably, theviral vector comprises a nucleic acid sequence of SEQ ID NO: 1, or asequence at least 90% identical thereto, operably linked to anexpression control sequence. Examples of suitable viral vectors includebut are not limited to adenoviral, retroviral, lentiviral, herpesvirusand adeno-associated virus (AAV) vectors.

In a preferred embodiment, the viral vector includes a portion of aparvovirus genome, such as an AAV genome with the rep and cap genesdeleted and/or replaced by the modified REP1 gene sequence and itsassociated expression control sequences. The modified human REP1 genesequence is typically inserted adjacent to one or two (i.e., is flankedby) AAV TRs or TR elements adequate for viral replication (Xiao et al.,1997, J. Virol. 71(2): 941-948), in place of the nucleic acid encodingviral rep and cap proteins. Other regulatory sequences suitable for usein facilitating tissue-specific expression of the modified REP1 genesequence in the target cell may also be included.

In some preferred embodiments, the AAV viral vector comprises a nucleicacid comprising from 5′ to 3′: (a) an AAV2 terminal repeat (b) a CAGpromoter (c) a codon optimized REP1 gene as herein described (d) apolyadenylation sequence and (e) an AAV2 terminal repeat. In aparticularly preferred embodiment, the AAV viral vector comprises anucleic acid (transgene cassette) comprising the sequence of SEQ ID NO:5or a sequence at least 90%, at least 95%, at least 98% or at least 99%identical thereto:

(SEQ ID NO: 5)TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC   60CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG AGAGGGAGTG  120GCCAACTCCA TCACTAGGGG TTCCTATCGA TTGAATTCCC CGGGGATCCA CTAGTTATTA  180ATAGTAATCA ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA  240ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT  300AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA  360GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTACGCC  420CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT  480ATGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGTCGA  540GGTGAGCCCC ACGTTCTGCT TCACTCTCCC CATCTCCCCC CCCTCCCCAC CCCCAATTTT  600GTATTTATTT ATTTTTTAAT TATTTTGTGC AGCGATGGGG GCGGGGGGGG GGGGGGGGCG  660CGCGCCAGGC GGGGCGGGGC GGGGCGAGGG GCGGGGCGGG GCGAGGCGGA GAGGTGCGGC  720GGCAGCCAAT CAGAGCGGCG CGCTCCGAAA GTTTCCTTTT ATGGCGAGGC GGCGGCGGCG  780GCGGCCCTAT AAAAAGCGAA GCGCGCGGCG GGCGGGGAGT CGCTGCGACG CTGCCTTCGC  840CCCGTGCCCC GCTCCGCCGC CGCCTCGCGC CGCCCGCCCC GGCTCTGACT GACCGCGTTA  900CTCCCACAGG TGAGCGGGCG GGACGGCCCT TCTCCTCCGG GCTGTAATTA GCGCTTGGTT  960TAATGACGGC TTGTTTCTTT TCTGTGGCTG CGTGAAAGCC TTGAGGGGCT CCGGGAGGGC 1020CCTTTGTGCG GGGGGAGCGG CTCGGGGGGT GCGTGCGTGT GTGTGTGCGT GGGGAGCGCC 1080GCGTGCGGCT CCGCGCTGCC CGGCGGCTGT GAGCGCTGCG GGCGCGGCGC GGGGCTTTGT 1140GCGCTCCGCA GTGTGCGCGA GGGGAGCGCG GCCGGGGGCG GTGCCCCGCG GTGCGGGGGG 1200GGCTGCGAGG GGAACAAAGG CTGCGTGCGG GGTGTGTGCG TGGGGGGGTG AGCAGGGGGT 1260GTGGGCGCGT CGGTCGGGCT GCAACCCCCC CTGCACCCCC CTCCCCGAGT TGCTGAGCAC 1320GGCCCGGCTT CGGGTGCGGG GCTCCGTACG GGGCGTGGCG CGGGGCTCGC CGTGCCGGGC 1380GGGGGGTGGC GGCAGGTGGG GGTGCCGGGC GGGGCGGGGC CGCCTCGGGC CGGGGAGGGC 1440TCGGGGGAGG GGCGCGGCGG CCCCCGGAGC GCCGGCGGCT GTCGAGGCGC GGCGAGCCGC 1500AGCCATTGCC TTTTATGGTA ATCGTGCGAG AGGGCGCAGG GACTTCCTTT GTCCCAAATC 1560TGTGCGGAGC CGAAATCTGG GAGGCGCCGC CGCACCCCCT CTAGCGGGCG CGGGGCGAAG 1620CGGTGCGGCG CCGGCAGGAA GGAAATGGGC GGGGAGGGCC TTCGTGCGTC GCCGCGCCGC 1680CGTCCCCTTC TCCCTCTCCA GCCTCGGGGC TGTCCGCGGG GGGACGGCTG CCTTCGGGGG 1740GGACGGGGCA GGGCGGGGTT CGGCTTCTGG CGTGTGACCG GCGGCTCTAG AGCCTCTGCT 1800AACCATGTTC ATGCCTTCTT CTTTTTCCTA CAGTCTAGAG TCGACCTGCA GAAGCTTCCA 1860CCATGGCTGA TACACTGCCT TCTGAGTTTG ATGTGATCGT GATTGGAACT GGACTGCCTG 1920AGAGTATTAT TGCTGCTGCT TGTAGTAGAA GCGGCCGGAG AGTGCTGCAC GTGGACAGCA 1980GATCCTACTA TGGCGGCAAC TGGGCCTCTT TCAGCTTTTC CGGCCTGCTG AGCTGGCTGA 2040AGGAGTACCA GGAGAACTCC GACATCGTGT CTGATAGCCC CGTGTGGCAG GACCAGATCC 2100TGGAGAATGA GGAGGCCATC GCCCTGTCCA GGAAGGATAA GACCATCCAG CACGTGGAGG 2160TGTTCTGCTA TGCCAGCCAG GACCTGCACG AGGATGTGGA GGAGGCAGGC GCCCTGCAGA 2220AGAACCACGC CCTGGTGACC TCCGCCAATT CTACAGAGGC CGCCGACTCC GCCTTTCTGC 2280CTACCGAGGA TGAGTCCCTG TCTACAATGT CTTGTGAGAT GCTGACCGAG CAGACACCTA 2340GCTCCGATCC AGAGAACGCC CTGGAGGTCA ATGGCGCCGA GGTGACCGGC GAGAAGGAGA 2400ACCACTGCGA CGATAAGACC TGCGTGCCAA GCACATCCGC CGAGGACATG TCCGAGAACG 2460TGCCTATCGC CGAGGATACC ACAGAGCAGC CAAAGAAGAA TCGCATCACA TACAGCCAGA 2520TCATCAAGGA GGGCAGGCGC TTCAATATCG ACCTGGTGTC TAAGCTGCTG TACAGCCGGG 2580GCCTGCTGAT CGATCTGCTG ATCAAGAGCA ACGTGTCCCG CTATGCCGAG TTCAAGAATA 2640TCACCAGAAT CCTGGCCTTT CGGGAGGGAA GAGTGGAGCA GGTGCCCTGC AGCAGAGCCG 2700ACGTGTTCAA CTCCAAGCAG CTGACAATGG TGGAGAAGAG GATGCTGATG AAGTTCCTGA 2760CATTTTGTAT GGAGTACGAG AAGTATCCAG ATGAGTACAA GGGCTATGAG GAGATCACCT 2820TTTACGAGTA TCTGAAGACC CAGAAGCTGA CACCCAATCT GCAGTACATC GTGATGCACT 2880CCATCGCCAT GACCTCTGAG ACAGCCTCTA GCACCATCGA CGGCCTGAAG GCCACAAAGA 2940ACTTCCTGCA CTGCCTGGGC CGGTACGGCA ATACACCCTT CCTGTTTCCT CTGTATGGCC 3000AGGGCGAGCT GCCCCAGTGC TTCTGTAGAA TGTGCGCCGT GTTTGGCGGC ATCTATTGCC 3060TGAGGCACTC TGTGCAGTGT CTGGTGGTGG ACAAGGAGAG CCGCAAGTGT AAGGCCATCA 3120TCGATCAGTT TGGCCAGCGG ATCATCTCTG AGCACTTCCT GGTGGAGGAC AGCTACTTTC 3180CTGAGAACAT GTGCTCCAGG GTGCAGTATC GCCAGATCAG CCGGGCCGTG CTGATCACCG 3240ATAGATCCGT GCTGAAGACA GACAGCGATC AGCAGATCAG CATCCTGACC GTGCCAGCAG 3300AGGAGCCAGG CACCTTCGCC GTGAGAGTGA TCGAGCTGTG CTCCTCTACC ATGACATGTA 3360TGAAGGGCAC CTACCTGGTG CACCTGACCT GCACAAGCTC CAAGACAGCC CGCGAGGACC 3420TGGAGAGCGT GGTGCAGAAG CTGTTCGTGC CCTACACCGA GATGGAGATC GAGAACGAGC 3480AGGTGGAGAA GCCTAGAATC CTGTGGGCCC TGTACTTCAA CATGAGAGAC TCTAGCGATA 3540TCTCTAGGAG CTGTTACAAC GATCTGCCCT CTAACGTGTA CGTGTGCAGC GGACCTGACT 3600GTGGCCTGGG AAACGATAAT GCCGTGAAGC AGGCCGAGAC ACTGTTCCAG GAGATTTGCC 3660CTAACGAGGA CTTTTGTCCC CCTCCACCCA ATCCAGAGGA TATCATCCTG GACGGCGATT 3720CCCTGCAGCC AGAGGCCTCT GAGTCCTCTG CCATCCCCGA GGCCAATAGC GAAACATTCA 3780AAGAAAGCAC AAATCTGGGA AACCTGGAAG AAAGTAGTGA GTAAGCCTCG AGCAGCGCTG 3840CTCGAGAGAT CTGCGGCCGC GAGCTCGGGG ATCCAGACAT GATAAGATAC ATTGATGAGT 3900TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG 3960CTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA 4020TTCATTTTAT GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC 4080TCTACAAATG TGGTATGGCT GATTATGATC AATGCATCCT AGCCGGAGGA ACCCCTAGTG 4140ATGGAGTTGG CCACTCCCTC TCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG 4200CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG 4260GGAGTGGCCA A 4271

The components of the transgene cassette of SEQ ID NO:5 and theirrespective locations are identified in Table 2 below:

TABLE 2 Location (bp) Component Length (bp)  1-145 5′ ITR 145  170-1833CAG promoter 1664 1863-3824 Codon-optimized hREP1 cDNA 1962 3867-4111SV40 PolyA 245 4127-4271 3′ ITR 145

The 5′ ITR has the following sequence:

(SEQ ID NO: 6) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

The 3′ ITR has the following sequence:

(SEQ ID NO: 7) AGCCGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG CCAA

The SV40 polyadenylation sequence has the following sequence:

(SEQ ID NO: 8) GAGCTCGGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTA TGATCAATGCATCCT

Those skilled in the art will appreciate that an AAV vector comprising atransgene and lacking virus proteins needed for viral replication (e.g.,cap and rep), cannot replicate since such proteins are necessary forvirus replication and packaging. Helper viruses include, typically,adenovirus or herpes simplex virus. Alternatively, as discussed below,the helper functions (E1a, E1b, E2a, E4, and VA RNA) can be provided toa packaging cell including by transfecting the cell with one or morenucleic acids encoding the various helper elements and/or the cell cancomprise the nucleic acid encoding the helper protein. For instance, HEK293 were generated by transforming human cells with adenovirus 5 DNA andnow express a number of adenoviral genes, including, but not limited toE1 and E3 (see, e.g., Graham et al., 1977, J. Gen. Virol. 36:59-72).Thus, those helper functions can be provided by the HEK 293 packagingcell without the need of supplying them to the cell by, e.g., a plasmidencoding them.

The viral vector may be any suitable nucleic acid construct, such as aDNA or RNA construct and may be single stranded, double stranded, orduplexed (i.e., self complementary as described in WO 2001/92551).

The viral capsid component of the packaged viral vectors may be aparvovirus capsid. AAV Cap and chimeric capsids are preferred. Forexample, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7 AAV8, AAV9, AAV10, AAV11, AAV12, AAV1.1, AAV2.5,AAV6.1, AAV6.3.1, AAV9.45, AAVrh10, AAVrh74, RHM4-1, AAV2-TT,AAV2-TT-S312N, AAV3B-S312N, AAV-LK03, snake AAV, avian AAV, bovine AAV,canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any otherAAV now known or later discovered. see, e.g., Fields et al., VIROLOGY,volume 2, chapter 69 (4.sup.th ed., Lippincott-Raven Publishers).

In some embodiments, the viral capsid component of the packaged viralvector is a variant of a native AAV capsid (i.e. comprises one or moremodifications relative to a native AAV capsid). In some embodiments, thecapsid is a variant of an AAV2, AAV5 or AAV8 capsid. In preferredembodiments, the capsid is a variant of an AAV2 capsid, such as thosedescribed in U.S. Patent Application Publication Number 2019/0255192A1(e.g. comprising the amino acid sequence of any of SEQ ID NOs: 42-59).In a particularly preferred embodiment, the capsid comprises a capsidprotein having the following amino acid sequence:

(SEQ ID NO: 9) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKAAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAISDQTKHARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

The variant AAV capsid protein of SEQ ID NO:9 contains the followingmodifications relative to native AAV2 capsid: (i) a proline (P) toalanine (A) mutation at amino acid position 34, which is located insidethe assembled capsid (VP1 protein only), and (ii) an insertion of 10amino acids (leucine-alanine-isoleucine-serine-asparticacid-glutamine-threonine-lysine-histidine-alanine/LAISDQTKHA) at aminoacid position 588, which is present in VP1, VP2, and VP3.

A full complement of AAV Cap proteins includes VP1, VP2, and VP3. TheORF comprising nucleotide sequences encoding AAV VP capsid proteins maycomprise less than a full complement AAV Cap proteins or the fullcomplement of AAV Cap proteins may be provided.

In yet another embodiment the present invention provides for the use ofancestral AAV vectors for use in therapeutic in vivo gene therapy.Specifically, in silico-derived sequences were synthesized de novo andcharacterized for biological activities. This effort led to thegeneration of nine functional putative ancestral AAVs and theidentification of Anc80, the predicted ancestor of AAV serotypes 1, 2, 8and 9 (Zinn et al., 2015, Cell Reports 12:1056-1068). Predicting andsynthesis of such ancestral sequences in addition to assembling into avirus particle may be accomplished by using the methods described in WO2015/054653, the contents of which are incorporated by reference herein.Notably, the use of the virus particles assembled from ancestral viralsequences may exhibit reduced susceptibility to pre-existing immunity incurrent day human population than do contemporary viruses or portionsthereof.

The invention includes packaging cells, which are encompassed by “hostcells,” which may be cultured to produce packaged viral vectors of theinvention. The packaging cells of the invention generally include cellswith heterologous (1) viral vector function(s), (2) packagingfunction(s), and (3) helper function(s). Each of these componentfunctions is discussed in the ensuing sections.

Initially, the vectors can be made by several methods known to skilledartisans (see, e.g., WO 2013/063379). A preferred method is described inGrieger, et al. 2015, Molecular Therapy 24(2):287-297, the contents ofwhich are incorporated by reference herein for all purposes. Briefly,efficient transfection of HEK293 cells is used as a starting point,wherein an adherent HEK293 cell line from a qualified clinical mastercell bank is used to grow in animal component-free suspension conditionsin shaker flasks and WAVE bioreactors that allow for rapid and scalablerAAV production. Using the triple transfection method (e.g., WO96/40240), the suspension HEK293 cell line generates greater than 10⁵vector genome containing particles (vg)/cell or greater than 10¹⁴ vg/Lof cell culture when harvested 48 hours post-transfection. Morespecifically, triple transfection refers to the fact that the packagingcell is transfected with three plasmids: one plasmid encodes the AAV repand cap genes, another plasmid encodes various helper functions (e.g.,adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VA RNA, andanother plasmid encodes the transgene and its various control elements(e.g., modified REP1 gene and CAG promoter).

To achieve the desired yields, a number of variables are optimized suchas selection of a compatible serum-free suspension media that supportsboth growth and transfection, selection of a transfection reagent,transfection conditions and cell density. A universal purificationstrategy, based on ion exchange chromatography methods, was alsodeveloped that resulted in high purity vector preps of AAV serotypes1-6, 8, 9 and various chimeric capsids. This user-friendly process canbe completed within one week, results in high full to empty particleratios (>90% full particles), provides post-purification yields (>10¹³vg/L) and purity suitable for clinical applications and is universalwith respect to all serotypes and chimeric particles. This scalablemanufacturing technology has been utilized to manufacture GMP Phase Iclinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B(scAAV8), Giant Axonal Neuropathy (scAAV9) and Retinitis Pigmentosa(AAV2), which have been administered into patients. In addition, aminimum of a 5-fold increase in overall vector production byimplementing a perfusion method that entails harvesting rAAV from theculture media at numerous time-points post-transfection.

The packaging cells include viral vector functions, along with packagingand vector functions. The viral vector functions typically include aportion of a parvovirus genome, such as an AAV genome, with rep and capdeleted and replaced by the modified REP1 sequence and its associatedexpression control sequences. The viral vector functions includesufficient expression control sequences to result in replication of theviral vector for packaging. Typically, the viral vector includes aportion of a parvovirus genome, such as an AAV genome with rep and capdeleted and replaced by the transgene and its associated expressioncontrol sequences. The transgene is typically flanked by two AAV TRs, inplace of the deleted viral rep and cap ORFs. Appropriate expressioncontrol sequences are included, such as a tissue-specific promoter andother regulatory sequences suitable for use in facilitatingtissue-specific expression of the transgene in the target cell. Thetransgene is typically a nucleic acid sequence that can be expressed toproduce a therapeutic polypeptide or a marker polypeptide.

The terminal repeats (TR(s)) (resolvable and non-resolvable) selectedfor use in the viral vectors are preferably AAV sequences, withserotypes 1, 2, 3, 4, 5 and 6 being preferred. Resolvable AAV TRs neednot have a wild-type TR sequence (e.g., a wild-type sequence may bealtered by insertion, deletion, truncation or missense mutations), aslong as the TR mediates the desired functions, e.g., virus packaging,integration, and/or provirus rescue, and the like. The TRs may besynthetic sequences that function as AAV inverted terminal repeats, suchas the “double-D sequence” as described in U.S. Pat. No. 5,478,745 toSamulski et al., the entire disclosure of which is incorporated in itsentirety herein by reference. Typically, but not necessarily, the TRsare from the same parvovirus, e.g., both TR sequences are from AAV2.

The packaging functions include capsid components. The capsid componentsare preferably from a parvoviral capsid, such as an AAV capsid or achimeric AAV capsid function. Examples of suitable parvovirus viralcapsid components are capsid components from the family Parvoviridae,such as an autonomous parvovirus or a Dependovirus. For example, thecapsid components may be selected from AAV capsids, e.g., AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10,AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6,AAV Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9,AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03, and othernovel capsids as yet unidentified or from non-human primate sources.Capsid components may include components from two or more AAV capsids.

The packaged viral vector generally includes the modified REP1 genesequence and expression control sequences flanked by TR elements,referred to herein as the “transgene” or “transgene expressioncassette,” sufficient to result in packaging of the vector DNA andsubsequent expression of the modified REP1 gene sequence in thetransduced cell. The viral vector functions may, for example, besupplied to the cell as a component of a plasmid or an amplicon. Theviral vector functions may exist extrachromosomally within the cell lineand/or may be integrated into the cell's chromosomal DNA.

Any method of introducing the nucleotide sequence carrying the viralvector functions into a cellular host for replication and packaging maybe employed, including but not limited to, electroporation, calciumphosphate precipitation, microinjection, cationic or anionic liposomes,and liposomes in combination with a nuclear localization signal. Inembodiments wherein the viral vector functions are provided bytransfection using a virus vector; standard methods for producing viralinfection may be used.

The packaging functions include genes for viral vector replication andpackaging. Thus, for example, the packaging functions may include, asneeded, functions necessary for viral gene expression, viral vectorreplication, rescue of the viral vector from the integrated state, viralgene expression, and packaging of the viral vector into a viralparticle. The packaging functions may be supplied together or separatelyto the packaging cell using a genetic construct such as a plasmid or anamplicon, a Baculovirus, or HSV helper construct. The packagingfunctions may exist extrachromosomally within the packaging cell, butare preferably integrated into the cell's chromosomal DNA. Examplesinclude genes encoding AAV Rep and Cap proteins.

The helper functions include helper virus elements needed forestablishing active infection of the packaging cell, which is requiredto initiate packaging of the viral vector. Examples include functionsderived from adenovirus, baculovirus and/or herpes virus sufficient toresult in packaging of the viral vector. For example, adenovirus helperfunctions will typically include adenovirus components E1a, E1 b, E2a,E4, and VA RNA. The packaging functions may be supplied by infection ofthe packaging cell with the required virus. The packaging functions maybe supplied together or separately to the packaging cell using a geneticconstruct such as a plasmid or an amplicon. See, e.g., pXR helperplasmids as described in Rabinowitz et al., 2002, J. Virol. 76:791, andpDG plasmids described in Grimm et al., 1998, Human Gene Therapy9:2745-2760. The packaging functions may exist extrachromosomally withinthe packaging cell, but are preferably integrated into the cell'schromosomal DNA (e.g., E1 or E3 in HEK 293 cells).

Any suitable helper virus functions may be employed. For example, wherethe packaging cells are insect cells, baculovirus may serve as a helpervirus. Herpes virus may also be used as a helper virus in AAV packagingmethods. Hybrid herpes viruses encoding the AAV Rep protein(s) mayadvantageously facilitate for more scalable AAV vector productionschemes.

Any method of introducing the nucleotide sequence carrying the helperfunctions into a cellular host for replication and packaging may beemployed, including but not limited to, electroporation, calciumphosphate precipitation, microinjection, cationic or anionic liposomes,and liposomes in combination with a nuclear localization signal. Inembodiments wherein the helper functions are provided by transfectionusing a virus vector or infection using a helper virus; standard methodsfor producing viral infection may be used.

Any suitable permissive or packaging cell known in the art may beemployed in the production of the packaged viral vector. Mammalian cellsor insect cells are preferred. Examples of cells useful for theproduction of packaging cells in the practice of the invention include,for example, human cell lines, such as VERO, W138, MRC5, A549, HEK 293cells (which express functional adenoviral E1 under the control of aconstitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2,HuH7, and HT1080 cell lines. In one aspect, the packaging cell iscapable of growing in suspension culture, more preferably, the cell iscapable of growing in serum-free culture. In one embodiment, thepackaging cell is a HEK293 that grows in suspension in serum freemedium. In another embodiment, the packaging cell is the HEK293 celldescribed in U.S. Pat. No. 9,441,206 and deposited as ATCC No. PTA13274. Numerous rAAV packaging cell lines are known in the art,including, but not limited to, those disclosed in WO 2002/46359. Inanother aspect, the packaging cell is cultured in the form of a cellstack (e.g. 10-layer cell stack seeded with HEK293 cells).

Cell lines for use as packaging cells include insect cell lines. Anyinsect cell which allows for replication of AAV and which can bemaintained in culture can be used in accordance with the presentinvention. Examples include Spodoptera frugiperda, such as the Sf9 orSf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines,e.g., Aedes albopictus derived cell lines. A preferred cell line is theSpodoptera frugiperda Sf9 cell line. The following references areincorporated herein for their teachings concerning use of insect cellsfor expression of heterologous polypeptides, methods of introducingnucleic acids into such cells, and methods of maintaining such cells inculture: Methods in Molecular Biology, ed. Richard, Humana Press, N J(1995); O'Reilly et al., Baculovirus Expression Vectors: A LaboratoryManual, Oxford Univ. Press (1994); Samulski et al., 1989, J. Virol.63:3822-3828; Kajigaya et al., 1991, Proc. Nat'l. Acad. Sci. USA 88:4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer etal., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; andSamulski et al., U.S. Pat. No. 6,204,059.

Virus capsids according to the invention can be produced using anymethod known in the art, e.g., by expression from a baculovirus (Brownet al., (1994) Virology 198:477-488). As a further alternative, thevirus vectors of the invention can be produced in insect cells usingbaculovirus vectors to deliver the rep/cap genes and rAAV template asdescribed, for example, by Urabe et al., 2002, Human Gene Therapy13:1935-1943.

In another aspect, the present invention provides for a method of rAAVproduction in insect cells wherein a baculovirus packaging system orvectors may be constructed to carry the AAV Rep and Cap coding region byengineering these genes into the polyhedrin coding region of abaculovirus vector and producing viral recombinants by transfection intoa host cell. Notably when using Baculavirus production for AAV,preferably the AAV DNA vector product is a self-complementary AAV likemolecule without using mutation to the AAV ITR. This appears to be aby-product of inefficient AAV rep nicking in insect cells which resultsin a self-complementary DNA molecule by virtue of lack of functional Repenzyme activity. The host cell is a baculovirus-infected cell or hasintroduced therein additional nucleic acid encoding baculovirus helperfunctions or includes these baculovirus helper functions therein. Thesebaculovirus viruses can express the AAV components and subsequentlyfacilitate the production of the capsids.

During production, the packaging cells generally include one or moreviral vector functions along with helper functions and packagingfunctions sufficient to result in replication and packaging of the viralvector. These various functions may be supplied together or separatelyto the packaging cell using a genetic construct such as a plasmid or anamplicon, and they may exist extrachromosomally within the cell line orintegrated into the cell's chromosomes.

The cells may be supplied with any one or more of the stated functionsalready incorporated, e.g., a cell line with one or more vectorfunctions incorporated extrachromosomally or integrated into the cell'schromosomal DNA, a cell line with one or more packaging functionsincorporated extrachromosomally or integrated into the cell'schromosomal DNA, or a cell line with helper functions incorporatedextrachromosomally or integrated into the cell's chromosomal DNA.

The rAAV vector may be purified by methods standard in the art such asby column chromatography or cesium chloride gradients. Methods forpurifying rAAV vectors are known in the art and include methodsdescribed in Clark et al., 1999, Human Gene Therapy 10(6):1031-1039;Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Pat. No.6,566,118 and WO 98/09657.

Treatment Methods

In certain embodiments, a method is provided for the treatment ofchoroideremia in a subject in need of such treatment by administering tothe subject a therapeutically effective amount of a nucleic acid havinga nucleotide sequence at least 90%, at least 95%, at least 98%identical, or 100% identical to the nucleotide sequence of SEQ ID NO:1or a pharmaceutical composition comprising such a nucleic acid and atleast one pharmaceutically acceptable excipient.

In related aspects, a nucleic acid comprising a nucleotide sequence atleast 90%, at least 95%, at least 98% identical or 100% identical to thenucleotide sequence of SEQ ID NO:1 for use in the treatment ofchoroideremia is provided.

In other related aspects, the use of a nucleic acid comprising anucleotide sequence at least 90%, at least 95%, at least 98% identicalor 100% identical to the nucleotide sequence of SEQ ID NO:1 for themanufacture of a medicament is provided.

In other related aspects, the use of a nucleic acid comprising anucleotide sequence at least 90%, at least 95%, at least 98% identicalor 100% identical to the nucleotide sequence of SEQ ID NO:1 for themanufacture of a medicament for the treatment of choroideremia isprovided.

In some aspects, the nucleotide sequence at least 90%, at least 95%, atleast 98% identical or 100% identical to the nucleotide sequence of SEQID NO:1 is operably linked to an expression control sequence. In someembodiments, the nucleotide sequence of SEQ ID NO:1 is operably linkedto a CAG promoter. In some preferred embodiments, the CAG promoter hasthe sequence of SEQ ID NO:4.

In some embodiments, the nucleotide sequence at least 90%, at least 95%,at least 98% identical or 100% identical to the nucleotide sequence ofSEQ ID NO:1 forms part of an expression cassette. In some aspects, theexpression cassette comprises from 5′ to 3′: (a) an AAV2 terminal repeat(b) a CAG promoter (c) codon optimized REP1 gene of SEQ ID NO:1 (d) anSV40 polyadenylation sequence and (e) an AAV2 terminal repeat. Inpreferred embodiments, the 5′ AAV2 terminal repeat has the nucleotidesequence set forth as SEQ ID NO:6 and/or the CAG promoter has thenucleotide sequence set forth as SEQ ID NO:4 and/or the SV40polyadenylation sequence has the nucleotide sequence set forth as SEQ IDNO:8 and/or the 3′ AAV2 terminal repeat has the nucleotide sequence setforth as SEQ ID NO:7. In a particularly preferred embodiment, theexpression cassette comprises a nucleic acid comprising the nucleotidesequence of SEQ ID NO:5 or a sequence at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identical thereto.

In further embodiments, a method is provided for the treatment ofchoroideremia in a subject in need of such treatment by administering tothe subject a therapeutically effective amount of a recombinant AAV(rAAV) virion, or a pharmaceutical composition comprising same, the rAAVvirion comprising (i) a nucleic acid having a nucleotide sequence atleast 90%, at least 95%, at least 98% identical or 100% identical to thenucleotide sequence of SEQ ID NO:1 operably linked to an expressioncontrol sequence and (ii) an AAV capsid.

In related embodiments, provided is the use of a recombinant AAV (rAAV)virion comprising (i) a nucleic acid having a nucleotide sequence atleast 90%, at least 95%, at least 98% identical or 100% identical to thenucleotide sequence of SEQ ID NO:1 operably linked to an expressioncontrol sequence and (ii) an AAV capsid for the treatment ofchoroideremia.

In other related embodiments, provided is the use of a recombinant AAV(rAAV) virion comprising (i) a nucleic acid having a nucleotide sequenceat least 90%, at least 95%, at least 98% identical or 100% identical tothe nucleotide sequence of SEQ ID NO:1 operably linked to an expressioncontrol sequence and (ii) an AAV capsid for the manufacture of amedicament for the treatment of choroideremia.

In some embodiments, the rAAV virion comprises a native AAV2, AAV4, AAV5or AAV8 capsid. In other embodiments, the rAAV virion comprises avariant AAV capsid that comprises one or more modifications relative toAAV2, AAV4, AAV5 or AAV8. In a preferred embodiment, the AAV capsidcomprises the sequence of SEQ ID NO:9.

In some embodiments, the rAAV virion comprises (i) a native AAV2 capsidor variant thereof and (ii) an expression cassette comprising from 5′ to3′: (a) an AAV2 terminal repeat (b) a CAG promoter (c) codon optimizedREP1 gene of SEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) anAAV2 terminal repeat. In preferred embodiments, the rAAV comprises (i) acapsid comprising a capsid protein of SEQ ID NO:9 and (ii) a nucleicacid comprising a 5′ AAV2 terminal repeat of SEQ ID NO:6, a CAG promoterof SEQ ID NO:4, an SV40 polyadenylation sequence of SEQ ID NO:8 and a 3′AAV2 terminal repeat of SEQ ID NO:7. In a particularly preferredembodiment, the rAAV comprises (i) a capsid comprising a capsid proteinof SEQ ID NO:9 and (ii) an expression cassette comprising the nucleotidesequence of SEQ ID NO:5.

In particularly preferred embodiments, the use of an rAAV in thetreatment of choroideremia or for the manufacture of a medicament forthe treatment of choroideremia is provided, wherein the rAAV comprises(i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO:5 and(ii) a capsid comprising a capsid protein having the amino acid sequenceof SEQ ID NO:9. In some aspects, the rAAV is administered byintravitreal injection.

In other particularly preferred embodiments, a method for the treatmentof choroideremia is provided comprising administering to the subject aneffective amount of an rAAV comprising (i) a nucleic acid comprising anucleotide sequence of SEQ ID NO:5 and (ii) a capsid comprising a capsidprotein having the amino acid sequence of SEQ ID NO:9. In some aspects,the rAAV is administered to the subject by intravitreal injection.

In other aspects, a pharmaceutical composition is provided comprising anucleic acid having a nucleotide sequence at least 90%, at least 95% atleast 98% identical or 100% identical to the nucleotide sequence of SEQID NO:1, optionally operably linked to an expression control sequence,and at least one pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises a nucleicacid comprising the nucleotide sequence of SEQ ID NO:1 operably linkedto a constitutive promoter, preferably a CAG promoter having a sequenceat least 90%, at least 95% at least 98% identical or 100% identical tothe nucleotide sequence of SEQ ID NO:4.

In other aspects, a pharmaceutical composition is provided comprising atleast one pharmaceutically acceptable excipient and an infectious rAAVcomprising (i) an AAV capsid and (ii) a nucleic acid comprising from 5′to 3′: (a) an AAV2 terminal repeat (b) a CAG promoter (c) codonoptimized REP1 gene of SEQ ID NO:1 (d) an SV40 polyadenylation sequenceand (e) an AAV2 terminal repeat. In related embodiments, thepharmaceutical composition comprises between 10⁹ vg and 10¹⁴ vg,preferably between 10¹⁰ vg and 10¹³ vg of the rAAV, more preferablycomprises about 3×10¹¹ vg or about 1×10¹² vg of the rAAV.

In preferred embodiments, the pharmaceutical composition comprises anrAAV comprising (i) a capsid comprising a capsid protein comprising orconsisting of the sequence of SEQ ID NO:9 and (ii) a nucleic acidcomprising codon optimized REP1 gene of SEQ ID NO:1, wherein the nucleicacid further comprises a 5′ AAV2 terminal repeat of SEQ ID NO:6 and/or aCAG promoter of SEQ ID NO:4 and/or an SV40 polyadenylation sequence ofSEQ ID NO:8 and/or an AAV2 terminal repeat of SEQ ID NO:7. In relatedembodiments, the pharmaceutical composition comprises between 10⁹ vg and10¹⁴ vg, preferably between 10¹⁰ vg and 10¹¹ vg of the rAAV, morepreferably comprises about 3×10¹¹ vg or about 1×10¹² vg of the rAAV.

In some embodiments, a method for expressing REP1 in one or more retinalpigmented epithelial cells and one or more rod photoreceptor cells of ahuman subject is provided comprising administering to the human subjectan effective amount of an infectious rAAV as herein described, whereinthe REP1 is expressed in the one or more retinal pigmented epithelialcells and one or more rod photoreceptor cells. In some preferredembodiments, the effective amount of infectious rAAV is 10⁹ vg/eye to10¹⁴ vg/eye and/or a single dose of the rAAV is intravitreallyadministered (bilaterally or unilaterally) to the human subject and/orthe rAAV comprises a capsid of SEQ ID NO:9 and/or the rAAV comprises aheterologous nucleic acid comprising the nucleotide sequence of SEQ IDNO:5.

In a particularly preferred embodiment, a pharmaceutical composition isprovided comprising at least one pharmaceutically acceptable excipientand an infectious rAAV comprising (i) a capsid comprising a capsidprotein comprising or consisting of the sequence of SEQ ID NO:9 and (ii)a nucleic acid comprising or consisting of the nucleotide sequence ofSEQ ID NO:5. In related embodiments, the pharmaceutical compositioncomprises 10⁹ vg and 10¹⁴ vg, preferably between 10¹⁰ vg and 10¹³ vg ofthe rAAV, more preferably comprises about 3×10¹¹ vg or about 1×10¹² vgof the rAAV.

In some embodiments, a nucleic acid or infectious rAAV as hereindescribed is administered by periocular or intraocular (intravitreal,suprachoroidal or subretinal) injection to a human with choroideremia,whereby the choroideremia is treated in the subject. In otherembodiments, a nucleic acid or infectious rAAV as herein described isadministered subretinally or intravitreally to a human withchoroideremia, whereby the choroideremia is treated in the subject. Inpreferred embodiments, a human subject with choroideremia isadministered a single intravitreal injection (bilateral or unilateral)of an rAAV as herein described.

In related aspects, treatment of choroideremia in a treated subjectcomprises (i) an improvement (i.e. gain) in visual function orfunctional vision relative to a control (e.g. relative to a baselinemeasurement in the treated patient prior to treatment, relative to theuntreated eye if the nucleic acid or rAAV is administered unilaterally,or relative to an untreated concurrent or historical control group ofchoroideremia patients) and/or (ii) a decrease in loss of visualfunction and/or retinal degeneration in a treated eye compared to acontrol (e.g. untreated eye in same patient or untreated control group)at e.g. 6 months, 12 months or 24 months after treatment. Theseimprovements can be assessed by an appropriate ophthalmological test,including but not limited to visual acuity testing, microperimetry andother visual field testing, anatomical testing, such as opticalcoherence tomography scans and fundus autofluorescence imaging, retinalelectrophysiology, and/or quality of life (QoL) assessments.

In some aspects, an effective amount of a nucleic acid or rAAV (orpharmaceutical composition comprising same) as herein described is anamount effective to treat choroideremia in a human patient. In relatedaspects, an effective amount of an rAAV as herein described is between10⁹ and 10¹⁴ rAAV particles (or vector genomes (vg))/eye), preferablybetween 10¹⁰ and 10¹³ vg/eye, or between 1×10¹¹ vg/eye and 5×10¹²vg/eye, more preferably is about 3×10¹¹ vg/eye or about 1×10¹² vg/eye.In some preferred embodiments, a single dose of about 3×10¹¹ vg/eye orabout 1×10¹² vg/eye is intravitreally administered to a human patientwith choroideremia, whereby the choroideremia is treated.

Some embodiments of the invention are exemplified in the following items1 to 41:

1. A nucleic acid encoding human Rab escort protein-1 (REP1) protein ofSEQ ID NO:2 and codon optimized for expression in humans, the nucleicacid comprising the nucleotide sequence set forth as SEQ ID NO: 1 orcomprising a nucleotide sequence at least 95% identical thereto, whereinthe nucleic acid is expressed at a greater level compared with the levelof expression of the wild type REP1 nucleotide sequence of SEQ ID NO: 3in an otherwise identical cell.

2. The nucleic acid according to item 1, wherein the nucleotide sequencehas a codon adaptation index of at least 0.94.

3. The nucleic acid according to item 1, comprising the nucleotidesequence set forth as SEQ ID NO: 1.

4. An expression cassette comprising the nucleic acid according to anyone of items 1 to 3 and an expression control sequence operably linkedand heterologous to the nucleic acid sequence.

5. The expression cassette of item 4, wherein the expression controlsequence is a constitutive promoter, preferably a CAG promotercomprising the nucleotide sequence set forth as SEQ ID NO:4 or asequence at least 90%, at least 95%, or at least 98% identical thereto.

6. The expression cassette of item 5, comprising from 5′ to 3′: (a) anAAV2 terminal repeat (b) a CAG promoter (c) codon optimized REP1 gene ofSEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) an AAV2terminal repeat.

7. The expression cassette of item 6, wherein the 5′ AAV2 terminalrepeat has the nucleotide sequence set forth as SEQ ID NO:6 and/orwherein the CAG promoter has the nucleotide sequence set forth as SEQ IDNO:4 and/or wherein the SV40 polyadenylation sequence has the nucleotidesequence set forth as SEQ ID NO:8 and/or wherein the 3′ AAV2 terminalrepeat has the nucleotide sequence set forth as SEQ ID NO:7.

8. The expression cassette of item 7, comprising or consisting of thenucleotide sequence of SEQ ID NO:5 or a sequence at least 90%, at least95%, at least 98% identical thereto.

9. The expression cassette of item 3, wherein the expression controlsequence is a promoter that directs preferential expression of thenucleic acid in rods and cones.

10. A vector comprising the nucleic acid according to any one of items 1to 3 or an expression cassette according to any one of items 4 to 9.

11. The vector of item 10, wherein the vector is a recombinantadeno-associated (rAAV) vector.

12. The vector of item 11, wherein the rAAV vector comprises an AAVcapsid of serotype 2, 4, 5 or 8 or a variant thereof.

13. The vector of item 12, wherein the rAAV vector comprises an AAV2capsid or variant thereof.

14. The vector of item 13, wherein the rAAV vector comprises an AAV2capsid variant comprising a capsid protein comprising or consisting ofthe sequence of SEQ ID NO:9.

15. The vector of any one of items 11-14, wherein the rAAV vectorcomprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminalrepeat (b) a CAG promoter (c) codon optimized REP1 gene of SEQ ID NO:1(d) an SV40 polyadenylation sequence and (e) an AAV2 terminal repeat.

16. The vector of item 15, wherein the 5′ AAV2 terminal repeat has thenucleotide sequence set forth as SEQ ID NO:6 and/or wherein the CAGpromoter has the nucleotide sequence set forth as SEQ ID NO:4 and/orwherein the SV40 polyadenylation sequence has the nucleotide sequenceset forth as SEQ ID NO:8 and/or wherein the 3′ AAV2 terminal repeat hasthe nucleotide sequence set forth as SEQ ID NO:7.

17. The vector of item 16, wherein the rAAV comprises a nucleic acidcomprising the nucleotide sequence of SEQ ID NO:5 or a sequence at least90%, at least 95% or at least 98% identical thereto.

18. The vector of item 17, wherein the rAAV comprises (i) a capsidcomprising a capsid protein comprising or consisting of the sequence ofSEQ ID NO:9 and (ii) a nucleic acid comprising or consisting of thenucleotide sequence of SEQ ID NO:5.

19. A host cell comprising the nucleic acid according to any one ofitems 1 to 3 or an expression cassette according to any one of items 4to 9.

20. The host cell according to item 19, wherein the host cell is amammalian cell.

21. The host cell of item 19 or 20, wherein the host cell is a CHO cell,an HEK293 cell, an HEK293T cell, a HeLa cell, a BHK21 cell or a Verocell and/or wherein the host cell is grown in a suspension or cell stackculture and/or wherein the host cell is a photoreceptor cell, a retinalganglion cell, a glial cell, a bipolar cell, an amacrine cell, ahorizontal cell, or a retinal pigmented epithelium cell.

22. A method for treating choroideremia in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a nucleic acid according to any one of items 1-3, anexpression cassette according to any one of items 4-9 or a vectoraccording to any one of items 10-18.

23. A method for treating choroideremia in a subject in need thereof,comprising administering to the subject an infectious rAAV comprising(i) an AAV capsid and (ii) a nucleic acid comprising from 5′ to 3′: (a)an AAV2 terminal repeat (b) a CAG promoter (c) codon optimized REP1 geneof SEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) an AAV2terminal repeat.

24. The method according to item 23, wherein the 5′ AAV2 terminal repeathas the nucleotide sequence set forth as SEQ ID NO:6 and/or wherein theCAG promoter has the nucleotide sequence set forth as SEQ ID NO:4 and/orwherein the SV40 polyadenylation sequence has the nucleotide sequenceset forth as SEQ ID NO:8 and/or wherein the 3′ AAV2 terminal repeat hasthe nucleotide sequence set forth as SEQ ID NO:7.

25. The method according to item 23 or 24, wherein the rAAV comprises(i) a capsid comprising a capsid protein comprising or consisting of thesequence of SEQ ID NO:9 and (ii) a nucleic acid comprising or consistingof the nucleotide sequence of SEQ ID NO:5.

26. The method according to any one of items 22-25, wherein the nucleicacid or vector is administered to the subject by intravitreal orsubretinal injection and/or wherein the vector is administered to thesubject at a dosage from about 10¹⁰ vector genomes (vg)/eye to about10¹³ vg/eye, preferably from about 1×10¹¹ vg/eye to about 5×10¹² vg/eye,more preferably at a dosage of about 3×10¹¹ vg/eye or at a dosage ofabout 1×10¹² vg/eye.

27. A nucleic acid according to any one of items 1-3, an expressioncassette according to any one of items 4-9, or a vector according to anyone of items 10-18 for use in the treatment of choroideremia.

28. A nucleic acid according to any one of items 1-3, an expressioncassette according to any one of items 4-9, or a vector according to anyone of items 10-18 for use in the manufacture of a medicament for thetreatment of choroideremia.

29. The nucleic acid, expression cassette or vector for use according toitem 27 or 28, wherein the nucleic acid or vector is administered byintravitreal or subretinal injection and/or wherein the vector is foradministration at a dosage from about 10¹⁰ vector genomes (vg)/eye toabout 10¹³ vg/eye, preferably from about 1×10¹¹ vg/eye to about 5×10¹²vg/eye, more preferably is for administration at a dosage of about3×10¹¹ vg/eye or at a dosage of about 1×10¹² vg/eye.

30. An infectious rAAV comprising (i) an AAV capsid and (ii) a nucleicacid comprising from 5′ to 3′: (a) an AAV2 terminal repeat (b) a CAGpromoter (c) codon optimized REP1 gene of SEQ ID NO:1 (d) an SV40polyadenylation sequence and (e) an AAV2 terminal repeat, for use in thetreatment of choroideremia.

31. An infectious rAAV comprising (i) an AAV capsid and (ii) a nucleicacid comprising from 5′ to 3′: (a) an AAV2 terminal repeat (b) a CAGpromoter (c) codon optimized REP1 gene of SEQ ID NO:1 (d) an SV40polyadenylation sequence and (e) an AAV2 terminal repeat, for use in themanufacture of a medicament for the treatment of choroideremia.

32 The infectious rAAV according to item 30 or 31, wherein the 5′ AAV2terminal repeat has the nucleotide sequence set forth as SEQ ID NO:6and/or wherein the CAG promoter has the nucleotide sequence set forth asSEQ ID NO:4 and/or wherein the SV40 polyadenylation sequence has thenucleotide sequence set forth as SEQ ID NO:8 and/or wherein the 3′ AAV2terminal repeat has the nucleotide sequence set forth as SEQ ID NO:7.

33. The infectious rAAV according to item 32, wherein the rAAV comprises(i) a capsid comprising a capsid protein comprising or consisting of thesequence of SEQ ID NO:9 and (ii) a nucleic acid comprising or consistingof the nucleotide sequence of SEQ ID NO:5.

34. The infectious rAAV for use according to any one of items 30-33,wherein the rAAV is administered by intravitreal injection and/orwherein the vector is administered at a dosage from about 10¹⁰ vectorgenomes (vg)/eye to about 10¹³ vg/eye, preferably from about 1×10¹¹vg/eye to about 5×10¹² vg/eye, more preferably is administered at adosage of about 3×10¹¹ vg/eye or at a dosage of about 1×10¹² vg/eye.

35. A method for treating a disease or condition mediated by a decreasedlevel of REP1 in a mammal, the method comprising administering atherapeutically effective amount of a nucleic acid according to any oneof items 1-3, an expression cassette according to any one of items 4-9,or a vector according to any one of items 10-18.

36. A method for increasing the level of REP1 in a mammal, the methodcomprising administering to the mammal a nucleic acid according to anyone of items 1-3, an expression cassette according to any one of items4-9, or a vector according to any one of items 10-18.

37. A pharmaceutical composition comprising a nucleic acid according toany one of items 1-3, an expression cassette according to any one ofitems 4-9, or a vector according to any one of items 10-18, and at leastone pharmaceutically acceptable excipient.

38. A pharmaceutical composition comprising an infectious rAAVcomprising (i) an AAV capsid and (ii) a nucleic acid comprising from 5′to 3′: (a) an AAV2 terminal repeat (b) a CAG promoter (c) codonoptimized REP1 gene of SEQ ID NO:1 (d) an SV40 polyadenylation sequenceand (e) an AAV2 terminal repeat.

39. The pharmaceutical composition according to item 38, wherein the 5′AAV2 terminal repeat has the nucleotide sequence set forth as SEQ IDNO:6 and/or wherein the CAG promoter has the nucleotide sequence setforth as SEQ ID NO:4 and/or wherein the SV40 polyadenylation sequencehas the nucleotide sequence set forth as SEQ ID NO:8 and/or wherein the3′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ IDNO:7.

40. The pharmaceutical composition according to item 39, wherein therAAV comprises (i) a capsid comprising a capsid protein comprising orconsisting of the sequence of SEQ ID NO:9 and (ii) a nucleic acidcomprising or consisting of the nucleotide sequence of SEQ ID NO:5.

41. The pharmaceutical composition according to any one of items 38-40,wherein the pharmaceutical composition comprises between 10⁹ vg and 10¹⁴vg of the rAAV, preferably between 10¹⁰ vg and 10¹³ vg of the rAAV, morepreferably comprises about 3×10¹¹ vg or about 1×10¹² vg of the rAAV.

EXAMPLES

The following examples illustrate preferred embodiments of the presentinvention and are not intended to limit the scope of the invention inany way. While this invention has been described in relation to itspreferred embodiments, various modifications thereof will be apparent toone skilled in the art from reading this application.

Example 1—Odon Optimization of REP cDNA Sequence

The human REP1 open reading frame cDNA sequence (NCBI Reference SequenceNM_000390.4) was codon optimized for human expression. The optimizationalgorithm included parameters including, but not limited to, codon usagebias, GC content, CpG dinucleotides content, negative CpG islands, mRNAsecondary structure, RNA instability motifs, cryptic splicing sites,premature polyadenylation sites, internal chi sites and ribosomalbinding sites, and repeat sequences.

The native human REP1 gene employs tandem rare codons that can reducethe efficiency of translation or even disengage the translationalmachinery. The codon usage bias in humans was changed by upgrading thecodon adaptation index (CAI) from 0.70 to 0.94. The average GC contentwas optimized from 54.24 in the native sequence to 61.22 in theoptimized sequence to prolong the half-life of the mRNA. Stem-Loopstructures, which impact ribosomal binding and stability of mRNA, werebroken in the optimized sequence. In addition, negative cis-acting sitessuch as ATTTA (6 of which are deleted in the optimized sequence) werescreened and deleted to optimize expression of the gene in human cellsand several restriction enzyme sites were deleted (2 BglII(AGATCT), 1EcoRI(GAATTC), 1 XhoI(CTCGAG) and 1 ARE sites were deleted).

The resulting codon optimized nucleotide sequence, set forth herein asSEQ ID NO:1, contains improved codon usage, altered GC content, bettermRNA stability, and modification of negative cis acting elementsrelative to the native sequence of SEQ ID NO:3.

Example 2—(Odon Ontimized REP1 eDNA Seauegce is Expressed at HigherLevels in RPE Cell. From Patients with Choroideremia

A human in vitro model system was generated to evaluate expression ofcodon optimized human REP1 nucleic acid having the nucleotide sequenceof SEQ ID NO:1 in diseased human RPE cells derived from humanchoroideremia patients and functional correction of the CHM diseasephenotype. This model system was chosen for in vitro pharmacologybecause a suitable non-human primate model of choroideremia (CHM) islacking for pre-clinical studies. Two CHM patient fibroblast sampleswere reprogrammed to iPSCs, then differentiated into functional matureRPE cells. Lack of Rep1 protein in CHM patients has been shown tocorrelate with cellular defects in Rab27a trafficking and prenylation(see e.g. Strunnikova, N. V. et al., PLoS Biol. 4, e8402 (2009);Sergeev, Y. V. et al., Mutat. Res.-Fundam. Mol. Mech. Mutagen, 665,44-50 (2009); Rak, A. et al., Cell 117, 749-760 (2004)).

Materials and Methods

Generation of Induced Pluripotent Stem Cell Lines from ChoroideremiaPatient Cells

Cellular reprogramming of fibroblasts from two choroideremia patients(referred to herein as CHM1 and CHM2), was performed by Simplicon RNAreprogramming (EMD Millipore). At day 10, approximately 5×10⁴-1×10⁵reprogrammed cells were re-plated on growth factor reduced Matrigel(Corning) in mouse embryonic fibroblasts (MEF)-conditioned mediumcontaining B18R protein (200 ng/mL) supplemented with human iPSCReprogramming Boost Supplement II (EMD Millipore). At day 20,reprogrammed cells, recognized by altered morphology and ability to formsmall colonies, were transitioned to mTeSR-1 media (Stem CellTechnologies). Colonies of approximately 200 cells or larger wereisolated manually and plated on growth factor reduced Matrigel coatedplates in mTeSR-1 medium. CHM-iPSC lines were expanded from a singlecolony. The CHM-iPSC lines were cultured on Vitronectin XF (Stem CellTechnologies) in mTeSR-1 maintenance medium and sub-cultured usingGentle Cell Dissociation Reagent (Stem Cell Technologies), every 4-5days at 70-80% confluence. To ensure random differentiation into allthree germ layers, iPSC embryoid bodies (EBs) were formed in suspensionculture for one week and then differentiated in adherent conditions foran additional four weeks in mTeSR-1 basal medium, plus 20% KnockoutSerum Replacement (Thermo Fisher Scientific).

Generation of Human Choroideremia Retinal Pigmented Epithelial (RPE)Cells

RPE cells were generated by a directed differentiation protocol aspreviously described (Leach et al., Investigative ophthalmology & visualscience 56(2):1002-13 (2015)). Briefly, iPSCs were passaged directlyonto Matrigel (BD Biosciences) in DMEM/F12 with 1×B27, 1×N2, and 1×NEAA(Invitrogen). From days 0 to 2, 50 ng/ml Noggin, 10 ng/ml Dkk1, 10 ng/mlIGF1 (R&D Systems Inc.), and 10 mM nicotinamide were added to the basemedium. From days 2 to 4, 10 ng/ml Noggin, 10 ng/ml Dkk1, 10 ng/ml IGF1,and 5 ng/ml bFGF and 10 mM nicotinamide were added to the base medium.From days 4 to 6, 10 ng/ml Dkk1 and 10 ng/ml IGF1 and in 100 ng/mlActivin A (R&D Systems) were added to the base medium. From days 6 to14, 100 ng/ml Activin A, 10 μM SU5402 (EMD Millipore), and 1 mM VIP(Sigma-Aldrich) were added to the base medium. At day 14, the cells weremechanically enriched by scraping away cells with non-RPE morphology.Subsequently, the remaining RPE were digested using TrypLE Express(Invitrogen) for ˜5 minutes at 37° C. The cells were passed through a30-μm single-cell strainer and seeded onto Matrigel-coated tissueculture plastic, transwell membranes (Corning Enterprises), orCC2-treated chambered slides in XVIVO-10 media (Lonza).

Functional Characterization of Human Choroideremia RPE Cells

CHM RPE cells were cultured using a formulated media to analyze rodouter segment (ROS) phagocytosis (Maminishkis, et al., InvestigativeOphthalmology and Visual Science, 47(8):3612-24 (2006)). Cells wereplated in quadruplicate at 1×10⁵ cells per cm² on 0.1% gelatin-coatedblack-walled, clear bottom 96 well plates and cultured for 30 days.Photoreceptor ROSs were isolated from bovine eyes (Sierra for MedicalScience) as previously described (Molday R S and Molday L L, Journal ofCell Biology, 105(6 Pt 1):2589-601 (1987)) and fluorescently labeledwith fluorescein isothiocyanate (FITC) protein (Thermo FisherScientific). In some conditions, cultured cells were treated with 62.5μg/ml αVβ5 integrin function-blocking antibody (Abcam) or IgG isotypecontrol (Abcam) for 30 minutes at 37° C. Following the initial antibodyincubation, cells were challenged with 1×10⁶ FITC-ROSs per well for fivehours at 37° C. and 5% CO2 (Buchholz et al., STEM CELLS TRANSLATIONALMEDICINE 2(5):384-93 (2013)) (Rowland et al., Journal of TissueEngineering and Regenerative Medicine, 7(8):642-53 (2013)). After ROSincubation, the wells were washed six times with PBS and 0.4% trypanblue was then added for 20 minutes to quench fluorescence fromextracellular ROS. Each well was imaged using epifluorescent microscopy,and integrated pixel density of photomicrographs was determined withImage J software using a rolling pixel radius of 50 (National Institutesof Health).

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde (PFA) (Santa CruzBiotechnologies) for 15 minutes at 4° C. All antibody staining was donein a blocking solution of PBS with 0.2% Triton-X100 (Sigma-Aldrich), 2%bovine serum albumin (Calbiochem), and 5% goat serum (Thermo Fisher).Primary antibody incubations were done overnight at 4° C. Cells werethen incubated with secondary antibodies for one hour at roomtemperature and then counterstained with DAPI (Sigma Aldrich) in PBS forfive minutes at room temperature. Cells were imaged using a Zeiss AxioObserver.D1. Image processing was performed using Zeiss Zen 2 softwareand FIJI. A list of primary and secondary antibodies is provided belowat Table 3:

TABLE 3 Antibody Host Company-Catalog No. Dilution Primary AntibodiesOCT4 Mouse Millipore-MAB4401 1:50  Nanog Rabbit Abcam-ab21624 1:50  SOX2Rabbit Abcam-ab92494 1:50  BEST1 Rabbit Abcam-ab14927 1:100 TUJ-1 MousePromega-G7121 1:200 HNF4-A Rabbit Santa Cruz-SC-8987 1:100 ASMA MouseSigma Aldrich-A2547 1:500 GFP Chicken Abcam-ab13970 1:200 MITF MouseThermo Fisher-MA5-14146 1:100 OTX2 Mouse R&D Systems-MAB1979 1:20 RPE-65 Rabbit Sant Cruz-sc32896 1:10  ZO-1 Mouse Thermo Fisher-33-91001:200 Secondary Antibodies Alexa Fluor488 Goat Invitrogen-A11078 1:250anti-rabbit Alexa Fluor555 Goat Invitrogen-A21428 1:250 anti-rabbitAlexa Fluor680 Goat Invitrogen-A21109 1:250 anti-rabbit Alexa Fluor488Goat Invitrogen-A11029 1:250 anti-mouse Alexa Fluor555 GoatInvitrogen-A21422 1:250 anti-mouse Alexa Fluor680 Goat Invitrogen-355181:250 anti-mouse

SDS-PAGE and Western Blot

CHM RPE cell lysates were harvested using a standard RIPA Buffer (ThermoFisher) with a Complete Protease Inhibitor Tablet (Millipore Sigma) andincubated on ice for 15 minutes. Samples were then centrifuged at 21×gfor 15 minutes. Supernatants were collected, and protein concentrationswere determined using a BCA protein assay (Thermo Fisher Scientific)normalized and adjusted to 2 μM DTT. Biorad 4× Sample Buffer was addedand samples were heated at 70° C. for 10 minutes. An XT Criterion gelwas run followed by gel transfer to a membrane. The membrane was thenblocked and probed with REP1 and GADPH antibodies. Membranes wereincubated with secondary antibodies conjugated to HRP and bands werevisualized with ECL.

Prenylation Assay

The prenylation assay was performed using RPE cell lysates as describedin Köhnke et al., PLoS ONE 8(12):1-11 (2013). Following a wash with PBS,cell lysates were prepared in cold Prenylation Buffer (500 μM HEPES pH7.0, 50 μM NaCl, 2 μM MgCl2, 0.1 μM GDP, 0.5% NP-40, and a CompleteProtease Inhibitor Tablet) and incubated on ice for 10-15 minutes.Protein concentrations were determined using a BCA protein assay (ThermoFisher Scientific). Protein concentrations were normalized, and lysatesadjusted to 2 μM DTT. Prenylation reactions were performed using 20 μLof lysate corresponding to 50-200 μg protein. The reaction for thefunctional complex was composed of 2 μM RabGGTase, 4 μM Rab27a and 4 μMBiotinGeranyl-PPi (Jena Bioscience). Reactions were incubated at 25° C.for 5 hours and stopped by adding 4× Sample Buffer (Biorad), DTT to 40mM and heating at 70° C. for 10 minutes. Western blotting was carriedout on XT Criterion gels according to manufacturer's protocols.Prenylation reactions were analyzed using streptavidin-HRP (Abcam).

Rab27A Trafficking Assay

RPE cells were seeded onto vitronectin coated eight chambered slides at25,000 cells per cm² in XVIVO-10 media. Two days after seeding, the CHMRPE cells were transduced with recombinant AAV virions comprising (i) atransgene expression cassette having the sequence of SEQ ID NO:5 and(ii) a modified AAV2 capsid protein having the amino acid sequence ofSEQ ID NO:9, at a multiplicity of infection (MOI) of 5000 vg/cell.Fourteen days post infection, cells were fixed and stained as describedabove.

Experimental Data

Two CHM patient fibroblast samples were obtained and reprogrammed toiPSCs followed by differentiation to RPE cells as described above. Morespecifically, cellular reprogramming of fibroblast cells (CHM1 and CHM2)was performed by Simplicon RNA reprogramming using synthetic in vitrotranscribed RNA expressing four reprogramming factors (Oct4, Klf4, Sox2and Glis1) in a polycistronic transcript that self-replicates for alimited number of cell divisions. Immunocytochemical analysis of humanPSC markers NANOG, SOX2 and OCT4 was performed to confirm thepluripotency of both choroideremia iPSC lines, CHM1 and CHM2 (FIGS. 1 aand 1 b ). To confirm pluripotency of the generated iPSC lines, cellswere randomly differentiated in suspension culture as EBs and thendifferentiated in adherent conditions for four weeks and evaluated forthe ability to differentiate into the ectodermal, mesodermal, andendodermal lineages. At that time, markers associated with neurons(TUJ1+), smooth muscle cells (ASMA+), and hepatocytes (HNF4A+) belongingto the ectoderm, mesoderm, and endoderm germ layers, respectively, weredetected (FIGS. 1 c and 1 d ). After confirmation of pluripotency, theiPSC lines generated from CHM1 and CHM2 patient cells weredifferentiated to RPE cells. RPE cells were allowed to mature for 30days, followed by analysis for proper RPE cell marker expression andfunction. Protein expression and localization of MelanogenesisAssociated Transcription Factor (MITF) and Orthodenticle Homeobox 2(OTX2), RPE65 and zonula occludens (ZO-1) (FIGS. 2 a and 2 b ) wasnormal. No changes in photoreceptor outer segment phagocytosis, a knownfunction of RPE, (FIG. 2 c ) confirmed that CHM iPSC-derived RPE cellsexhibit key physiological characteristics similar to those of nativeRPE.

Expression levels of codon optimized REP1 transgene versus unmodifiedREP1 were assessed. To that end, recombinant AAV (rAAV) virions wereisolated comprising (i) a modified AAV2 capsid protein having the aminoacid sequence of SEQ ID NO:9 and (ii) a transgene expression cassettecomprising either codon optimized REP1 of SEQ ID NO:1 or native REP1 ofSEQ ID NO:3, each under the control of a CAG promoter of SEQ ID NO:4.Briefly, CHM RPE cells were transduced with the rAAV virions at twodifferent MOIs, 500 or 5000 vg/cell. Cell lysates were collected 14 dayspost transduction and SDS-PAGE and Western blot analysis was carried outto evaluate REP1 expression levels. As illustrated in FIGS. 3A-B, codonoptimized REP1 resulted in higher expression levels than unmodifiedREP1. Further, REP1 protein levels reached levels found in normal RPE atthe lower dose (MOI 500 vg/cell).

A functional assay was developed to assess the ability of delivered REP1protein to prenylate Rab27a GTPase (FIG. 4 ). CHM RPE cells weretransduced with rAAV comprising (i) a transgene expression cassettehaving the sequence of SEQ ID NO:5 and (ii) a modified AAV2 capsidprotein having the amino acid sequence of SEQ ID NO:9. Cell lysates fromtransduced or control CHM1 and CHM2 RPE cells were collected 14 dayspost infection. Wild type RPE cells were used as a positive control.Prenylation of Rab27a GTPase will only occur in the presence of REP1and, the prenyl donor, RabGGTase. To visualize the prenyl transfer fromRabGGTase to Rab27a GTPase, the prenyl groups were labeled with biotin.The cell lysates, following transduction, were combined with Rab27aGTPase, RabGGTase and biotinylated prenyl groups. The in vitro reactionwas incubated for 5 hours to optimize prenyl group transfer. Followingthe reaction, the lysates were subjected to SDS-PAGE and Westernblotting analysis. A SA-HRP conjugate revealed the level of prenylationin each reaction (FIGS. 5 a-d ). RPE cells derived from a normalfibroblast cell-derived iPSC line were used as a positive control inthis experiment.

A second functional experiment was done to confirm that prenylation ofRab27a GTPases, following delivery of the rAAV results in propertrafficking of Rab27a to the target membrane. CHM RPE cells werecultivated at low density (2.5×10⁴ cells/cm²) and then transduced withrAAV comprising (i) a transgene expression cassette having the sequenceof SEQ ID NO:5 and (ii) a modified AAV2 capsid protein having the aminoacid sequence of SEQ ID NO:9 at a MOI of 5000 vg/cell. After 14 days,cultures were immunostained with anti-REP1 and anti-RAB27A antibodiesand imaged to visualize the subcellular localization of RAB27A intransduced versus untreated cultures. Treatment of CHM RPE cells (FIG. 6a ) with the rAAV caused trafficking of RAB27A from the cytoplasmicregions to target membranes (FIG. 6 b ) analogous to normalFB-iPSC-derived RPE cells (FIG. 6 c ). These data demonstrate that twoweeks after delivery of codon optimized REP1 of SEQ ID NO:1, RAB27Atrafficking from the cytoplasmic regions to target membranes wasnormalized and this correction was associated with restitution of thenormal cellular RPE phenotype.

Conclusion

The studies described above demonstrate that codon optimized REP1 of SEQID NO:1 is expressed at significantly higher levels in disease-relevant(REP1 deficient) human RPE cells compared to the native (unmodified)REP1 gene. The studies also demonstrate that REP1 expressed from codonoptimized REP1 of SEQ ID NO:1 is functional, rescues the prenylationdefect (Rab27) and corrects the intracellular trafficking defect in RAbproteins, thus restoring the normal cellular RPE phenotype in thediseased RPE cells. In vitro pharmacology indicates that cohREP1 showssuperior correction of REP1 protein deficiency in Retinal PigmentEpithelial (RPE) cells derived from choroideremia patients when comparedwith the normal gene.

Example 3—Assessment of Safety and Biodistribution of Codon OptimizedREP1 cDNA Sequence Delivered by R100 Via Intravitreal Administration inNon-Human Primates

Materials and Methods

GLP Toxicology and Biodistribution Studies

Male cynomolgus macaques (Macaca fascicularis) aged 2-14 years weredosed via two 50 μL intravitreal injections into each eye through thesclera for a total dose volume of 100 μL/eye. Doses of 1×10¹¹ vg/eye(unilateral administration), 3×10¹¹ vg/eye (bilateral administrationonly), and 1×10¹² vg/eye (unilateral & bilateral administration) wereevaluated. The animals were anesthetized with Ketamine IM and giventopical ophthalmic solutions to eliminate pain. 20-80 mg ofmethylprednisolone was administered by IM injection weeklypost-injection. Euthanasia was performed by trained veterinary staff atWeek 3, Week 13, and Week 26 post-administration.

4D-110 (rAAV comprising a capsid protein of SEQ ID NO:9 and aheterologous nucleic acid comprising the nucleotide sequence of SEQ IDNO:5) genome biodistribution was assessed in all major ocularcompartments (retina, optic nerve, ciliary body, iris, trabecularmeshwork), and major systemic organs (including the testes) usingvalidated, GLP-compliant qPCR assay. In tissues where genomes weredetected, transgene expression was assessed by a qualified,GLP-compliant RT-qPCR assay.

Serial toxicology assessments performed in the study were: clinicalocular evaluations (complete ophthalmic examinations, including SD-OCTimaging and ERG), systemic evaluations, clinical pathology, grosspathology and microscopic pathology. Assays were validated to determinethe anti-capsid and anti-transgene antibody responses. ELISpot assayswere validated to detect cellular responses to the R100 capsid(comprising a variant capsid protein of SEQ ID NO:9) and expressedproteins.

Neutralizing Antibody Assay

2v6.11 cells were plated at a density of 3×10⁴ cells/well 24 hours priorto infection. rAAV vectors encoding firefly luciferase driven by the CAGpromoter were incubated at 37° C. for 1 hour with individual serumsamples prior to infection, and cells were then infected at a genomicMOI of 1,000. Luciferase activity was assessed 48 hours post infectionusing the Luc-Screen Extended-Glow Luciferase Reporter Gene Assay System(Invitrogen) or the ONE-Glo Luciferase Assay System (Promega) andquantified using the BioTek Cytation 3 Cell Imaging Multi-Mode Readerand Gen5 software.

Prior to enrollment in studies, non-human primates (NHP) serum wasscreened for the presence of neutralizing antibodies against R100. NHPswere enrolled in studies when samples resulted in less than 50%neutralization of AAV transduction at a 1:10 serum dilution.

AAV Manufacturing

Recombinant R100 viral vectors were produced by transient transfectionin HEK293 cells. Cells were cultured in DMEM supplemented with FBS andwere maintained at 37° C. in a 5% CO₂ environment. Cells were triplytransfected (payload, capsid, and helper plasmids) usingpolyethylenimine (PEI). 48-96 hours post-transfection, viral particleswere harvested from cells and/or supernatant and cells lysed viamicrofluidization. Cell lysate and/or supernatant was enzymaticallytreated to degrade plasmid and host-cell DNA, then clarified andconcentrated by tangential flow filtration (TFF). The TFF retentate wasthen loaded onto an affinity resin column for purification. FollowingpH-gradient elution, post-affinity material was buffer exchanged, thenfurther purified (if needed) by anion-exchange chromatography. PurifiedrAAV was then formulated into DPBS with 0.001% polysorbate-20, sterilefiltered, and filled to yield rAAV Drug Product.

Results

4D-110 Delivery is Safe and Results in Expression of TherapeuticTransgene in NHP

4D-110 (R100.CAG-cohRep1) has been advanced into a Phase 1-2 clinicaltrial. Investigational New Drug (IND)-enabling data for this productincludes evaluation in two separate 6-month Good Laboratory Practices(GLP) toxicology and biodistribution studies (Table 4). A total of 61eyes of 44 NHPs were injected by intravitreal injection with either asingle eye administration, sequential bilateral administration, orsimultaneous bilateral administration.

TABLE 4 Good Laboratory Practices (GLP) Toxicology and BiodistributionStudies 4DMT Study Number Lot Number Number Gender Eye(s) Dose In-Life4D17-02 N/A 1 Male OD vehicle  3 weeks 4DEP000003.01 4 Male OD 1E+11vg/eye 4DEP000004.01 5 Male OD 1E+12 vg/eye N/A 1 Male OD vehicle 13weeks 4DEP000003.01 4 Male OD 1E+11 vg/eye 4DEP000004.01 5 Male OD 1E+12vg/eye N/A 1 Male OD vehicle 26 weeks 4DEP000003.01 4 Male OD 1E+11vg/eye 4DE1000004.01 5 Male OD 1E+12 vg/eye 4D18-13 N/A 3 Male OUvehicle 26 weeks 3 Male OU 1E+11 vg/eye 3 Male OD + OS 1E+11 vg/eye4DEP000011.01 4 Male OU 1E+12 vg/eye 4 Male OD + OS 1E+12 vg/eye 3 MaleOU 1E+12 vg/eye 13 weeks

No significant toxicities were observed with 4D-110 at either doselevel, as determined by clinical observations, histopathology, OCT, orERG. Administration of 4D-110 into a single eye resulted in only minimalto mild anterior uveitis that was restricted to the immediatepost-administration period and resolved by Week 3 (FIG. 9 ); in somecases systemic steroid doses were transiently increased. Bilateraladministration of 4D-110 resulted in transient minimal to moderateanterior uveitis in both low and high dose groups; this finding resolvedwithin two weeks generally, coincident with an increase in systemicsteroid treatment.

Very high levels of vector genomes were present in the retina of thetreated eye at all timepoints (week 3, left panel; week 13, middlepanel; week 26, right panel) indicating persistence of the vector inocular tissue (FIG. 10 ). In addition to the retina, vector genomes weredetected in the treated eye within samples from the aqueous humor,vitreous humor, iris/ciliary body, and the optic nerve at alltimepoints. Non-ocular tissues generally had no detectable vectorgenomes with the exception of low levels in liver, spleen, and the lymphnodes (FIG. 10 ). R100 vector-derived transgene expression was detectedin the treated retina and iris/ciliary body from both low and high dosegroups (FIG. 11 ). Gene expression was dose-dependent and increased fromWeek 3 to Week 13 and remained stable at Week 26 (FIG. 11 , left, middleand right panel respectively). No non-ocular vector expression wasdetected at Week 26 in any study (FIG. 11 ).

Using an ELISpot assay to evaluate cellular immune responses, no animalsdeveloped significant responses to R100 capsid peptides or transgenepeptides (data not shown). A majority of animals dosed with 4D-110generated an anti-capsid antibody response post-administration (data notshown).

Summary

4D-110 (R100.CAG-cohRep1) has recently been translated into a clinicaltrial for the inherited retinal disease choroideremia (NCT04483440).This therapeutic product has been evaluated in two separate GLPtoxicology and biodistribution studies (Table 4). A total of 44 NHPswere injected with a single eye administration, sequential bilateraladministration, or simultaneous bilateral administration; a total of 61NHP eyes were injected. No significant test-article-related adverseevents or T-cell responses were reported. Mild to moderate, transientcorticosteroid-responsive anterior uveitis was observed. Transgeneexpression was localized to the retina, and expression was not detectedin any of the systemic organs evaluated. Human clinical trials areunderway in order to determine the safety, pharmacodynamics, andefficacy (including through serial visual field testing and opticalcoherence tomography scans) of this product by intravitreal injection.

Example 4—Assessment of Safety of Codon Optimized REP1 eDNA SequenceDelivered by R100 Via Intravitreal Administration in Human ChoroideremiaPatients

Initial Phase 1 Dose Escalation Safety and Tolerability Data Summary

Clinical Trial Designs and Enrollment

The clinical trial employed a standard “3+3” dose-escalation designed toassess the safety, tolerability and biologic activity of a singleintravitreal injection of 4D-110 at two dose levels (3E11 or 1E12vg/eye). A total of six patients were enrolled across dose escalationcohorts, with three at each dose level. Patients received a standardimmunosuppression regimen with taper; adjustments were determined byinvestigators. The results described are based on data cut-offs between1-9 months post-administration.

Initial Tolerability and Adverse Event Profile

4D-110 was well-tolerated throughout the assessment period as outlinedin the treatment-emergent adverse event (AE) summary table (Table 5):

TABLE 5 Adverse Event Summary Patient # enrolled 6 Doses 3E11 or 1E12vg/eye Follow-up data cut-off (months) 1-9 months Dose-LimitingToxicities (DLTs) 0 (0%) Serious AE 0 (0%) Any CTCAE Grade ≥ 3 0 (0%)Retinal AE (Any Grade) 0 (0%) Uveitis CTCAE Grade 2 (moderate) 1/6 (17%)Uveitis CTCAE Grade 1 (mild) 4/6 (67%)

Clinical Assessments

Patients' ocular and systemic status is closely monitored includingdetailed ophthalmic evaluations and retinal imaging together with bloodtesting and systemic examinations, as necessary. A variety of visualfunction and anatomical assessments is performed to detect anypreliminary efficacy signal. These assessments include, but are notlimited to, measurements of ellipsoid zone (EZ) area, fundusautofluorescence, microperimetry, static automated perimetry, and bestcorrected visual acuity (BCVA).

While the materials and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the method described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention.

The invention claimed is:
 1. A nucleic acid encoding human Rab escortprotein-1 (REP1) protein of SEQ ID NO:2 and codon optimized forexpression in humans, the nucleic acid comprising the nucleotidesequence set forth as SEQ ID NO: 1 or comprising a nucleotide sequenceat least 90% identical thereto.
 2. The nucleic acid according to claim1, wherein the nucleotide sequence has a codon adaptation index of atleast 0.94.
 3. An expression cassette comprising the nucleic acidaccording to claim 1, wherein said nucleotide sequence is operablylinked to an expression control sequence.
 4. The expression cassette ofclaim 3, wherein the expression control sequence comprises aconstitutive promoter or comprises a promoter that directs cell-specificexpression of the nucleic acid in rod and cone cells.
 5. The expressioncassette of claim 4, wherein the expression control sequence comprises aCAG promoter.
 6. The expression cassette of claim 4, comprising from 5′to 3′: (a) a 5′ AAV2 terminal repeat (b) a CAG promoter (c) a nucleotidesequence at least 90% identical to the nucleotide sequence set forth inSEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) a 3′ AAV2terminal repeat.
 7. The expression cassette of claim 6, wherein the 5′AAV2 terminal repeat has the nucleotide sequence set forth as SEQ IDNO:6 and/or wherein the CAG promoter has the nucleotide sequence setforth as SEQ ID NO:4 and/or wherein the SV40 polyadenylation sequencehas the nucleotide sequence set forth as SEQ ID NO:8 and/or wherein the3′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ IDNO:7.
 8. A recombinant adeno-associated virus (rAAV) vector comprising aheterologous nucleic acid comprising the expression cassette accordingto claim
 3. 9. The rAAV vector of claim 8, wherein the rAAV vectorcomprises an AAV capsid of serotype 2, 4, 5 or 8 or a variant thereof.10. The rAAV vector of claim 9, wherein the rAAV vector comprises avariant AAV capsid protein comprising the amino acid sequence of SEQ IDNO:9.
 11. The rAAV vector of claim 10, wherein the rAAV vector comprisesa nucleic acid comprising from 5′ to 3′: (a) a 5′ AAV2 terminal repeat(b) a CAG promoter (c) a nucleotide sequence at least 90% identical tothe nucleotide sequence set forth in SEQ ID NO:1 (d) an SV40polyadenylation sequence and (e) a 3′ AAV2 terminal repeat.
 12. The rAAVvector of claim 11, wherein the 5′ AAV2 terminal repeat has thenucleotide sequence set forth as SEQ ID NO:6 and/or wherein the CAGpromoter has the nucleotide sequence set forth as SEQ ID NO:4 and/orwherein the SV40 polyadenylation sequence has the nucleotide sequenceset forth as SEQ ID NO:8 and/or wherein the 3′ AAV2 terminal repeat hasthe nucleotide sequence set forth as SEQ ID NO:7.
 13. A method fortreating choroideremia in a human subject in need thereof, comprisingadministering to the subject a therapeutically effective amount ofpharmaceutical composition comprising an rAAV vector according to claim8 and a pharmaceutically acceptable excipient, whereby the choroideremiais treated in the human subject.
 14. The method according to claim 13,wherein the rAAV vector comprises a nucleic acid comprising from 5′ to3′: (a) a 5′ AAV2 terminal repeat (b) a CAG promoter (c) a nucleotidesequence at least 90% identical to the nucleotide sequence set forth inSEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) a 3′ AAV2terminal repeat.
 15. The method according to claim 14, wherein the 5′AAV2 terminal repeat has the nucleotide sequence set forth as SEQ IDNO:6 and/or wherein the CAG promoter has the nucleotide sequence setforth as SEQ ID NO:4 and/or wherein the SV40 polyadenylation sequencehas the nucleotide sequence set forth as SEQ ID NO:8 and/or wherein the3′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ IDNO:7.
 16. The method according to claim 13, wherein the rAAV vectorcomprises an AAV capsid of serotype 2, 4, 5 or 8 or a variant thereof.17. The method according to claim 16, wherein the rAAV vector comprisesa variant AAV capsid protein comprising the amino acid sequence of SEQID NO:9.
 18. The method according to claim 13, wherein thepharmaceutical composition is administered to the subject by periocular,intravitreal, suprachoroidal or subretinal injection.
 19. The methodaccording to claim 18, wherein the vector is administered to the subjectat a dosage from about 10¹⁰ vector genomes (vg)/eye to about 10¹³vg/eye.
 20. A pharmaceutical composition comprising the rAAV vectoraccording to claim 8 and at least one pharmaceutically acceptableexcipient.